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.

Transcription factor TBX18 promotes adult rat bone mesenchymal stem cell differentiation to biological pacemaker cells.

Bone mesenchymal stem cells (BMSCs) are currently considered the optimal stem cells for biological pacemaker cell transformation. The cardiac­specific transcription factor T­Box protein 18 (TBX18) is essential for sinoatrial node (SAN) formation, particularly formation of the head region that generates the electrical impulses that induce heart contraction. The present study aimed to confirm the effects of TBX18 on biological pacemaker differentiation of rat BMSCs. Flow cytometry was used to identify the surface markers of BMSCs, in order to acquire pure mesenchymal stem cells. Subsequently, BMSCs were transduced with TBX18 or green fluorescent protein adenovirus vectors. The effects of TBX18 were evaluated using SAN­specific makers including TBX18, α­actin, cardiac troponin I, hyperpolarization­activated cyclic nucleotide­gated channel 4 and connexin 43 by reverse transcription­quantitative polymerase chain reaction, western blotting and immunofluorescence. The findings demonstrated that direct conversion of BMSCs to biological pacemaker cells via TBX18 is a feasible method in the field of cardiology.

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.

Development of the cardiac conduction system in zebrafish.

The cardiac conduction system (CCS) propagates and coordinates the electrical excitation that originates from the pacemaker cells, throughout the heart, resulting in rhythmic heartbeat. Its defects result in life-threatening arrhythmias and sudden cardiac death. Understanding of the factors involved in the formation and function of the CCS remains incomplete. By transposon assisted transgenesis, we have developed enhancer trap (ET) lines of zebrafish that express fluorescent protein in the pacemaker cells at the sino-atrial node (SAN) and the atrio-ventricular region (AVR), termed CCS transgenics. This expression pattern begins at the stage when the heart undergoes looping morphogenesis at 36 h post fertilization (hpf) and is maintained into adulthood. Using the CCS transgenics, we investigated the effects of perturbation of cardiac function, as simulated by either the absence of endothelium or hemodynamic stimulation, on the cardiac conduction cells, which resulted in abnormal compaction of the SAN. To uncover the identity of the gene represented by the EGFP expression in the CCS transgenics, we mapped the transposon integration sites on the zebrafish genome to positions in close proximity to the gene encoding fibroblast growth homologous factor 2a (fhf2a). Fhf2a is represented by three transcripts, one of which is expressed in the developing heart. These transgenics are useful tools for studies of development of the CCS and cardiac disease.

Phosphorylation of Shox2 is required for its function to control sinoatrial node formation.

BACKGROUND: Inactivation of Shox2, a member of the short-stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. METHODS AND RESULTS: A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2-hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p-Shox2a) in cell culture as well as in the developing mouse and human SAN. Site-directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal-regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a-S92AS110A, mutant in the developing heart resulted in down-regulation of Nkx2.5 in wild-type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. CONCLUSIONS: Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.

Gene regulatory networks in cardiac conduction system development.

The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Given its critical role in coordinating cardiac performance, a detailed analysis of the molecular mechanisms underlying conduction system formation should inform our understanding of arrhythmia pathophysiology and affect the development of novel therapeutic strategies. Historically, the ability to distinguish cells of the conduction system from neighboring working myocytes presented a major technical challenge for performing comprehensive mechanistic studies. Early lineage tracing experiments suggested that conduction cells derive from cardiomyocyte precursors, and these claims have been substantiated by using more contemporary approaches. However, regional specialization of conduction cells adds an additional layer of complexity to this system, and it appears that different components of the conduction system utilize unique modes of developmental formation. The identification of numerous transcription factors and their downstream target genes involved in regional differentiation of the conduction system has provided insight into how lineage commitment is achieved. Furthermore, by adopting cutting-edge genetic techniques in combination with sophisticated phenotyping capabilities, investigators have made substantial progress in delineating the regulatory networks that orchestrate conduction system formation and their role in cardiac rhythm and physiology. This review describes the connectivity of these gene regulatory networks in cardiac conduction system development and discusses how they provide a foundation for understanding normal and pathological human cardiac rhythms.

Changes in the expression of ion channels, connexins and Ca2+-handling proteins in the sino-atrial node during postnatal development.

There are important postnatal changes in the sino-atrial node (SAN), the pacemaker of the heart. Compared with the neonate, the adult has a slower intrinsic heart rate and a longer SAN action potential. These changes may be due to differences in ion channel expression. Consequently, we investigated postnatal developmental changes in the expression of ion channels and Ca(2+)-handling proteins in the SAN to see whether this is indeed the case. Using quantitative PCR, in situ hybridization and immunohistochemistry, we investigated the expression of ion channels, Ca(2+)-handling proteins and connexins in the SAN from neonatal (2-7 days of age) and adult (∼6 months of age) New Zealand White rabbits. The spontaneous beating rate of adult SAN preparations was 21% slower than that of neonatal preparations. During postnatal development, quantitative PCR revealed a significant decline in the SAN of the following mRNAs: HCN4 (major isoform responsible for I(f)), Na(V)1.5 (responsible for I(Na)), Ca(V)1.3 (in part responsible for I(Ca,L)) and NCX1 (responsible for inward I(NaCa)). These declines could be responsible for the slowing of the pacemaker during postnatal development. There was a significant decline during development in mRNA for delayed rectifier K(+) channel subunits (K(V)1.5, responsible for I(K,ur), K(V)LQT1 and minK, responsible for I(K,s), and ERG, responsible for I(K,r)) and this could explain the prolongation of the action potential. In situ hybridization confirmed the changes observed by quantitative PCR. In addition, immunohistochemistry revealed hypertrophy of nodal cells during postnatal development. Moreover, there were complex changes in the expression of Ca(2+)-handling proteins with age. In summary, there are significant postnatal changes in the expression of ion channels and Ca(2+)-handling proteins in the SAN that could explain the established changes in heart rate and action potential duration that occur during normal development.

Porcine left atrial and sinoatrial 5-HT(4) receptor-induced responses: fading of the response and influence of development.

1.--In this study, we aimed to characterize in vitro the effects of the benzofuran 5-HT(4) receptor agonists prucalopride, R149402 and R199715 and the indolic agents tegaserod and 5-HT in the atria of young pigs (10-11 weeks) and newborn piglets. 2.--In the paced left atrium of young pigs, only 5-HT results in positive inotropic responses when administered cumulatively (maximal effect relative to isoprenaline=53%, pEC(50)=6.8); however, all agonists showed lusitropic effects. Noncumulative administration results in greater positive inotropic responses for 5-HT and induces moderate positive inotropic responses for the other agonists; these responses fade. 3.--Phosphodiesterase (PDE) enzyme inhibition with 3-isobutyl-1-methylxanthine (IBMX; 20 microM) enhances the responses to cumulatively administered 5-HT (maximal effect=89%, pEC(50)=7.7) and reveals clear positive inotropic effects for prucalopride, tegaserod, R149402 and R199715; fading is abolished. The maximal effect of the benzofurans is less pronounced than that of the indoles. 4.--In the spontaneously beating right atrium of young pigs, all agonists show chronotropic activity when administered cumulatively in the absence of IBMX, without fade. Benzofurans behaved as partial agonists compared to 5-HT (maximal effect=54%, pEC(50)=6.5). 5.--In newborns, the inotropic activity of the agonists in the IBMX-treated left atrium was less pronounced than in the young pig; the same applied for the chronotropic response in the right atrium, except for 5-HT. 6.--In conclusion, the atrial responses to 5-HT(4) receptor activation increase in the first months of life; the inotropic response is regulated by PDEs. Prucalopride, R149402 and R199715 are partial agonists compared to 5-HT.

Developmental changes in sinus node function in growing children: an updated analysis.

Sinus node dysfunction (SAND) may be congenital or acquired following injury or surgery for congenital heart lesions. Sinus node function is evaluated by electrophysiological (EP) parameters of corrected sinus node recovery time (CSNRT) and sinoatrial conduction time (SACT). The aim of this study was to determine age- and gender-specific values for CSNRT and SACT in pediatric patients without structural congenital heart disease. Data were collected on 152 patients who underwent an EP study for evaluation of supraventricular tachycardia between 1997 and 2002. All patients received midazolam and propofol and/or isoflurane for sedation and anesthesia, which are known to not affect EP parameters. The age of transition at which CSNRT changed significantly was 14 years (241.5 +/- 102.0 msec in patients younger than 14 years old and 285.6 +/- 144.3 msec in those older than 14 years, p < 0.05). The upper limits of normal CSNRT (mean + 2 SD) were significantly higher (445 vs 275 msec) and the upper limits of normal SACT values were lower (120 vs 200 msec) than the currently used norms in the younger age group. CSNRT values and atrial refractory period values were significantly longer in males compared to age-matched females [278.5 +/- 15.3 VS : 236.4 +/- 13.6 msec (p < 0.05) and 269.0 +/- 4.9 VS: 244.7 +/- 6.8 msec (p < 0.005), respectively]. The new age- and gender-specific values of EP parameters, which reflect sinus node function, may enable more precise recognition of SAND.

L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node.

Although the neonatal sinus node beats at a faster rate than the adult, when a sodium current (I(Na)) present in the newborn is blocked, the spontaneous rate is slower in neonatal myocytes than in adult myocytes. This suggests a possible functional substitution of I(Na) by another current during development. We used ruptured [T-type calcium current (I(Ca,T))] and perforated [L-type calcium current (I(Ca,L))] patch clamps to study developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. I(Ca,T) density did not differ with age, and no significant differences were found in the voltage dependence of activation or inactivation. I(Ca,L) density was lower in the adult than newborn (12.1 +/- 1.4 vs. 17.6 +/- 2.5 pA/pF, P = 0.049). However, activation and inactivation midpoints were shifted in opposite directions, reducing the potential contribution during late diastolic depolarization in the newborn (activation midpoints -17.3 +/- 0.8 and -22.3 +/- 1.4 mV in the newborn and adult, respectively, P = 0.001; inactivation midpoints -33.4 +/- 1.4 and -28.3 +/- 1.7 mV for the newborn and adult, respectively, P = 0.038). Recovery of I(Ca,L) from inactivation was also slower in the newborn. The results suggest that a smaller but more negatively activating and rapidly recovering I(Ca,L) in the adult sinus node may contribute to the enhanced impulse initiation at this age in the absence of I(Na).

Properties and modulation of If in newborn versus adult cardiac SA node.

The hearts in newborn mammals have greater intrinsic beating rates, rates of diastolic depolarization, and sensitivity to autonomic stimulation than those in adults. The differences could be explained partly by altered properties of the hyperpolarization-activated current (If). To test this possibility, sinoatrial node myocytes from the hearts of newborn (9- to 10-day) and adult (>30-day) rabbits were isolated, and the If was examined with the perforated-patch-clamp technique. The fully activated current-voltage relationship yielded a larger slope conductance of If in newborn SA node myocytes (0.244 +/- 0.020 vs. 0.158 +/- 0.012 pS/pF), compatible with the more rapid diastolic depolarization. Activation curves of the If had similar midactivation voltages (newborn, -66.71 +/- 1.94 mV; adult, -66.33 +/- 2.60 mV), but the slope was significantly greater in newborns (inverse slope factor: newborn, -9.57 +/- 0.35 mV; adult, -11.34 +/- 0.54 mV). No differences in shifts of the If activation curve in response to maximal concentrations of acetylcholine (newborn, -9.70 +/- 1.8 mV; adult, -12.60 +/- 2.10 mV) and isoproterenol (newborn, 6.90 +/- 2.5 mV; adult, 5.3 +/- 1.5 mV) or in the total shift in response to these agonists (newborn, 16.60 +/- 3.30 mV; adult, 18.00 +/- 1.00 mV) were observed. The greater If density and steeper voltage dependence can contribute to both the greater heart rate and the greater sensitivity of the SA node to autonomic modulation in newborn animals.

Postnatal maturation of the response of the canine sinus node to critically timed, brief vagal stimulation.

We have previously demonstrated that vagal phase-response curves (PRC), which characterize the effects of critically timed, brief vagal stimuli on sinus node automaticity, exhibit a fundamentally different shape in the canine newborn than in the adult. In this study we analyzed the changes in sinus cycle length in response to critically timed, brief vagal stimuli, delivered to the decentralized cervical right and left vagosympathetic trunks, in two older age groups: 14 1-mo-old puppies (ages 21-36 d), and eight 2-mo-old puppies (ages 56-62 d). Vagal PRC were constructed by plotting the magnitude (percent change) of the vagal chronotropic response as a function of the phase of the cardiac cycle at which the vagus nerve was stimulated. At 1 mo of age adult-type PRC were observed, but in only six of the puppies (43%) and only in response to right vagal stimulation. By 2 mo of age adult-type PRC were observed in seven of eight puppies (88%) in response to right vagal stimulation and in three of eight (38%) in response to left vagal stimulation. Thus, clear developmental changes in the phase dependence of the vagal chronotropic response can be tracked over the first 2 mo of life in the dog.

Quantitative histological analysis of the human sinoatrial node during growth and aging.

BACKGROUND: Fibrosis or fatty infiltration of the human sinoatrial (SA) node is generally believed to represent replacement of the SA nodal cells by connective tissue. Quantitative analysis, however, has not been performed precisely to validate the interpretation of such histological changes. METHODS AND RESULTS: The actual volume of the SA node and its components were calculated according to the sum of the pixel number representing the colors of SA nodal cells and connective tissue in serial sections using a digital color image analyzer. Average volume occupied by the total SA nodal cells in adolescents and adults (n = 7) was 3.55 +/- 0.45 mm3, which was 2.4 times greater than that in infants (n = 3). The rate of increase was smaller than that of the total SA node (4.2 times, 16.68 +/- 2.56 mm3 in adolescents and adults). The considerable discrepancy in the growth ratio between the SA nodal cells and the total SA node resulted from an increase in the volume of connective tissue (7.4 times). In the elderly (n = 9), the volume of total SA node and SA nodal cells actually decreased (13.10 +/- 1.85 mm3 and 2.18 +/- 0.44 mm3), whereas that of fibrous connective tissue remained unchanged. Constant DNA ploidy patterns of SA nodal cells determined by cytofluorometry indicated that SA nodal cells never synthesize DNA during growth. CONCLUSIONS: Until adulthood, the actual volume of SA nodal cells does not decrease, although the increase in volume ratio of the interstitial tissue to the total SA node has merely given a false impression of involution of SA nodal cells. Atrophy of SA nodal cells, however, occurs during aging together with reduction of the SA node and/or infiltration of fatty tissue.

Morphological innervation pattern of the developing rabbit heart.

The morphological innervation pattern of developing fetal and neonatal rabbit hearts was delineated histochemically by a cholinesterase/silver procedure and immunohistochemically with the monoclonal antibody HNK1, an antibody which recognizes some cells derived from neuroectoderm. Cholinesterase-containing nerves appeared distally on the outflow tract by gestational day 15 (G15). Isolated cells with cholinesterase-stained fine processes were present near the base of the pulmonary trunk. HNK1 antibody stained the same nerves and ganglia revealed by the cholinesterase reaction and other nerves in the rabbit heart. It was used to confirm that cells with fine neuron-like processes were present before nerve ingrowth. The G14 heart contained many HNK1 staining cells in the right atrium, outflow, and inflow tracts; cells with fine processes were few but increased at G16. By G17, a plexus of interweaving nerves and associated cells began to form at the base of the pulmonary trunk. Fine nerves encircled the base of the aorta, and others crossed the intercaval region dorsally. At G19, nerves 1) extended downward from a rich "bulbar" plexus along the front ventricular surface, 2) grew near the epicardial surface at the base of the heart along the atrial floor and ventricular roof, 3) traversed the vena cavae and intercaval region to enter the atrial roof, and 4) crossed the coronary sinus to reach the back ventricular walls. By G23, cholinesterase-staining nerves and ganglia in the atria and, epicardially, in the ventricles formed the general innervation pattern of the newborn and adult rabbit heart.

Postnatal development of chronotropic responses to chemical and electrical stimulation of adrenergic nerve endings in the sinoatrial node of the heart of the albino rat.

The positive chronotropic response to stimulation of adrenergic nerve endings in the sinoatrial node was studied in isolated atria from the hearts of rats of different ages. Dimethylphenylpiperazinium (DMPP) was used for chemical stimulation and transmural stimulation of the sinoatrial node region as electrical stimulation; in both cases noradrenaline is released from the nerve endings. With both stimulation methods, postnatal development was recorded in two phases. In the first phase, positive chronotropic responses are markedly increased and attained the maximum at the age of 14 days on using DMPP and of 24 days on using electrical stimulation. In the second phase, positive chronotropic responses diminish and at the age of about 45 days, with both stimulation methods, they become reduced to adult level. The first developmental phase can be attributed to an increase in the noradrenaline content of the nerve endings and the release of a larger amount of the transmitter during stimulation, together with an increase in the noradrenaline sensitivity of the cells of the sinoatrial node. It is not clear why positive chronotropic responses decrease in the second phase, when the noradrenaline content of the myocardial tissue continues to rise and pacemaker sensitivity to noradrenaline is not reduced.

Ultrastructural quantitative characterisation of sinus and atrioventricular nodal cells in the developing caprine heart compared with ordinary atrial and ventricular myocardial cells.

Cellular growth and changes in subcellular components as well as in intercellular junction structure were investigated quantitatively in sinus and atrioventricular nodes in comparison with right atrial and left ventricular myocardial cells of the goat heart in fetuses aged 6, 8, 10, 12, 14, 16 and 18 gestational weeks, young animals aged 1 and 3 postnatal weeks, and adults. At the earliest stage examined (sixth gestational week) no clear difference was recognised, in the patterns of star diagrams representing cellular characteristics, between nodal cells and ordinary cardiac muscle cells, except for a larger number of myofibrils in the ordinary cells. Atrial specific granules also appeared in the atrial cells at this stage. In general, initial signs of differentiation in ordinary cardiac muscle cells were detected at the sixteenth gestational week when transverse tubule formation occurred in the ventricular muscle cells. Throughout the developmental period, no appreciable alterations in the ultrastructural features were found in the sinus nodal cells whereas the atrioventricular nodal cells revealed slight changes at the later fetal and adult stages.

Developmental changes in the rabbit sinus node action potential and its response to adrenergic agonists.

We used standard microelectrode techniques to study developmental changes in the transmembrane potentials of sinus nodes from 1- to 10-day-old and adult rabbits superfused with Tyrode's solution at 37 degrees C. Maximum diastolic potential did not change with age, but action potential amplitude in day 1 to 3 hearts was lower than that in adults. Sinus cycle length (SCL) also was shorter in the young sinus nodes. In experiments on beta adrenergic stimulation the reduction of SCL in the young occurred at lower isoproterenol concentrations than in the adult, but the maximum reduction in SCL was the same at both ages (-40%). The alpha agonist, phenylephrine, had no age-dependent effect on SCL or action potential characteristics in either age group. In addition, the inhibitory effect of alpha stimulation demonstrated previously in automatic fibers of the ventricular specialized conducting system and atrium did not occur in sinus node. Studies of Purkinje fibers demonstrated this inhibitory effect to be voltage-dependent; it did not occur at membrane potentials in the range of those in the sinus node. Our results suggest that whereas developmental changes in the response to beta adrenergic stimulation are demonstrable in the sinus node, there are no such changes in the response to alpha adrenergic stimulation.

Progressive postnatal changes in sinus node response to atropine and propranolol.

Postnatal development of autonomic control of heart rate was evaluated in the sinus nodes of isolated, perfused right atria obtained from 21 sibling puppies in three different litters between 3 and 11 wk postpartum. Age-related changes in response to autonomic blockade indicated that propranolol administered after atropine had its most profound direct depressive effect on less mature atrial cells. In the youngest sinus nodes the familial antimuscarinic action (increase in sinus rate) of atropine was regularly preceded by a brief period of cholinomimetic action (a marked slowing of sinus rate) in the youngest sinus nodes. These two opposing effects of atropine underwent a developmental change during the 2-mo study. The cholinomimetic action diminished, whereas the antimuscarinic action increased as a function of age.