Ionic current changes underlying action potential repolarization responses to physiological pacing and adrenergic stimulation in adult rat ventricular myocytes.

This study aimed to simulate ventricular responses to elevations in myocyte pacing and adrenergic stimulation using a novel electrophysiological rat model and investigate ion channel responses underlying action potential (AP) modulations. Peak ion currents and AP repolarization to 50% and 90% of full repolarization (APD50-90 ) were recorded during simulations at 1-10 Hz pacing under control and adrenergic stimulation conditions. Further simulations were performed with incremental ion current block (L-type calcium current, ICa ; transient outward current, Ito ; slow delayed rectifier potassium current, IKs ; rapid delayed rectifier potassium current, IKr ; inward rectifier potassium current, IK1 ) to identify current influence on AP response to exercise. Simulated APD50-90 closely resembled experimental findings. Rate-dependent increases in IKs (6%-101%), IKr (141%-1339%), and ICa (0%-15%) and reductions in Ito (11%-57%) and IK1 (1%-9%) were observed. Meanwhile, adrenergic stimulation triggered moderate increases in all currents (23%-67%) except IK1 . Further analyses suggest AP plateau is most sensitive to modulations in Ito and ICa while late repolarization is most sensitive to IK1 , ICa , and IKs , with alterations in IKs predominantly stimulating the greatest magnitude of influence on late repolarization (35%-846% APD90 prolongation). The modified Leeds rat model (mLR) is capable of accurately modeling APs during physiological stress. This study highlights the importance of ICa , Ito , IK1, and IKs in controlling electrophysiological responses to exercise. This work will benefit the study of cardiac dysfunction, arrythmia, and disease, though future physiologically relevant experimental studies and model development are required.

Pharmacy and Exercise as Complimentary Partners for Successful Cardiovascular Ageing.

Diseases of the cardiovascular system have been the biggest cause of mortality for the majority of the last century, currently contributing to almost a third of deaths every year globally. Ageing associates with changes to the structure and function of the heart and vascular system that progressively increase the incidence of abnormalities, morbidity, and cardiovascular disease. The burden of ageing and its relationship to cardiovascular disease risk highlights the need for more research into the underlying mechanisms involved and how they may be treated and/or prevented. Factors influencing adrenergic dysfunction may explain a significant part of the age-related deterioration in health and responsiveness of the cardiovascular system. Increased sympathetic activity in old age overstimulates adrenergic receptors and causes detrimental changes within the associated signalling mechanisms, including a reduction in receptor number and downstream effector efficiency. Pharmacological agents, such as metformin, resveratrol, beta-blockers, and angiotensin converting enzyme (ACE) inhibitors, have been identified as potential anti-ageing therapies with cardiovascular effects, which may be beneficial in treating the decline in cardiovascular function with old age. Regular exercise has also shown promise in the prevention and treatment of harmful age-related effects on the cardiovascular system. This review will investigate age-associated vascular and cardiac remodelling, and the link between adrenergic dysfunction and vascular and cardiac control. This review will also consider whether pharmacological or non-pharmacological therapies are most effective, or indeed complimentary to potentially optimised ageing of the cardiovascular system and improved quality of life in the elderly.

Action potential responses to changes in stimulation frequency and isoproterenol in rat ventricular myocytes.

PURPOSE: Current understanding of ventricular action potential adaptation to physiological stress is generally based on protocols using non-physiological rates and conditions isolating rate effects from escalating adrenergic stimulation. To permit refined understanding, ventricular action potentials were assessed across physiological pacing frequencies in the presence and absence of adrenergic stimuli. Isolated and combined effects were analyzed to assess their ability to replicate in-vivo responses. METHODS: Steady-state action potentials from ventricular myocytes isolated from male Wistar rats (3 months; N = 8 animals) were recorded at 37°C with steady-state pacing at 1, 2, 4, 6, 8 and 10 Hz using whole-cell patch-clamp. Action potential repolarization to 25, 50, 75, 90 and 100% of full repolarization (APD25-100 ) was compared before and after 5 nM, 100 nM and 1 µM isoproterenol doses. RESULTS: A Repeated measures ANOVA found APD50-90  shortened with 5 nM isoproterenol infusion by 6-25% (but comparable across doses) (p ≤ 0.03). Pacing frequencies emulating a normal rat heart rate (6 Hz) prolonged APD50 23% compared with 1 Hz pacing. Frequencies emulating exercise or stress (10 Hz) shortened APD90 (29%). CONCLUSION: These results demonstrate modest action potential shortening in response to adrenergic stimulation and elevations in pacing beyond physiological resting rates. Our findings indicate changes in action potential plateau and late repolarization predominantly underlie simulated exercise responses in the rat heart. This work provides novel action potential reference data and will help model cardiac responses to physiological stimuli in the rat heart via computational techniques.

Reduced cardiac response to the adrenergic system is a key limiting factor for physical capacity in old age.

Ageing is associated with a progressive reduction in physical capacity reducing quality of life. One key physiological limitation of physical capacity that deteriorates in a progressive age-dependent manner is cardiac reserve. Peak cardiac output falls progressively with advancing age such that in extreme old age there is limited ability to enhance cardiac output beyond basal function as is required to support the increased metabolic needs of physical activity. This loss of dynamic range in cardiac output associates with a progressive reduction in the heart's response to adrenergic stimulation. A combination of decreases in the expression and functioning of beta1 adrenergic receptors partially underlies this change. Changes in end effector proteins also have a role to play in this decline. Alterations in the efficiency of excitation-contraction coupling contribute to the reduced chronotropic, inotropic and lusitropic responses of the aged heart. Moderate to vigorous endurance exercise training however has some potential to counter elements of these changes. Further studies are required to fully elucidate the key pivotal mechanisms involved in the age-related loss of response to adrenergic signalling to allow targeted therapeutic strategies to be developed with the aim of preserving physical capacity in advanced old age.

K2p 3.1 protein is expressed as a transmural gradient across the rat left ventricular free wall.

INTRODUCTION: K2p 3.1, also known as TASK-1, is a twin-pore acid-sensitive repolarizing K+ channel, responsible for a background potassium current that significantly contributes to setting the resting membrane potential of cardiac myocytes. Inhibition of IK2p3.1 alters cardiac repolarization and is proarrhythmogenic. In this study, we have examined the expression of K2p 3.1 and function of this channel in tissue and myocytes from across the left ventricular free wall. METHODS AND RESULTS: Using fluorescence immunocytochemistry, the expression of K2p 3.1 protein in myocytes from the subendocardial region was found to be twice (205% ± 13.5%) that found in myocytes from the subepicardial region of the left ventricle (100% ± 5.3%). The left ventricular free wall exhibited a marked transmural gradient of K2p 3.1 protein expression. Western blot analysis confirmed significantly higher K2p 3.1 protein expression in subendocardial tissue (156% ± 2.5%) than subepicardial tissue (100% ± 5.0%). However, there was no difference in K2p 3.1 messenger RNA expression. Whole-cell patch clamp identified IK2p3.1 current density to be significantly greater in myocytes isolated from the subendocardium (7.66 ± 0.53 pA/pF) compared with those from the subepicardium (3.47 ± 0.74 pA/pF). CONCLUSIONS: This is the first study to identify a transmural gradient of K2p 3.1 in the left ventricle. This gradient has implications for understanding ventricular arrhythmogenesis under conditions of ischemia but also in response to other modulatory factors, such as adrenergic stimulation and the presence of anesthetics that inhibits or activates this channel.

Interactions of Short-Term and Chronic Treadmill Training With Aging of the Left Ventricle of the Heart.

With aging, there is a decline in cardiac function accompanying increasing risk of arrhythmias. These effects are likely to be mechanistically associated with age-associated changes in calcium regulation within cardiac myocytes. Previous studies suggest that lifelong exercise can potentially reduce age-associated changes in the heart. Although exercise itself is associated with changes in cardiac function, little is known about the interactions of aging and exercise with respect to myocyte calcium regulation. To investigate this, adult (12 months) and old (24 months) C57/Bl6 mice were trained using moderate-intensity treadmill running. In response to 10 weeks' training, comparable cardiac hypertrophic responses were observed, although aging independently associated with additional cardiac hypertrophy. Old animals also showed increased L- and T-type calcium channels, the sodium-calcium exchange, sarcoendoplasmic reticulum calcium ATPase, and collagen (by 50%, 92%, 66%, 88%, and 113% respectively). Short-term exercise training increased D-type and T-type calcium channels in old animals only, whereas an increase in sodium-calcium exchange was seen only in adult animals. Long-term (12 months) training generally opposed the effects of aging. Significant hypertrophy remained in long-term trained old animals, but levels of sarcoendoplasmic reticulum calcium ATPase, sodium-calcium exchange, and collagen were not significantly different from those found in the adult trained animals.

Progressive age-associated activation of JNK associates with conduction disruption in the aged atrium.

Connexin43 (Cx43) is critical for maintaining electrical conduction across atrial muscle. During progressive ageing atrial conduction slows associating with increasing susceptibility to arrhythmias. Changes in Cx43 protein expression, or its phosphorylation status, can instigate changes in the conduction of the cardiac action potential. This study investigated whether increased levels of activated c-jun N-terminal kinase (JNK) is responsible for the decline of Cx43 during ageing. Right atria from guinea pigs aged between 1 day and 38 months of age were examined. The area of the intercalated disc increased with age concurrent with a 75% decline in C43 protein expression. An age-dependent increase in activated-JNK correlated with a rise in phosphorylated Cx43, but also slowing of action potential conduction velocity across the atria from 0.38±0.01 m/s at 1 month of age to 0.30±0.01 m/s at 38 months. The JNK activator anisomycin increased activated JNK in myocytes and reduced Cx43 protein expression simulating ageing. The JNK inhibitor SP600125, was found to eradicate almost all trace of Cx43 protein. We conclude that in vivo activation of JNK increases with age leading to the loss of Cx43 protein resulting in impaired conduction and contributing to the increasing risk of atrial arrhythmias with advancing age.

Aging is a primary risk factor for cardiac arrhythmias: disruption of intracellular Ca2+ regulation as a key suspect.

Aging is an inevitable time-dependent progression associated with a functional decline of the cardiovascular system even in 'healthy' individuals. Age positively correlates with an increasing risk of cardiac problems including arrhythmias. Not only the prevalence but also the severity of arrhythmias escalates with age. The reasons for this are multifactorial but dysregulation of intracellular calcium within the heart is likely to play a key role in initiating and perpetuating these life-threatening events. We now know that several aspects of cardiac calcium regulation significantly change with advancing age - changes that could produce electrical instability. Further development of knowledge of the mechanisms underlying these changes will allow us to reduce what currently is an inevitable increase in the incidence of arrhythmias in the elderly.

Distinguishing properties of cells from the myocardial sleeves of the pulmonary veins: a comparison of normal and abnormal pacemakers.

BACKGROUND: A common source of arrhythmogenic spontaneous activity instigating atrial fibrillation is the myocardial tissue, or sleeves, at the base of the pulmonary veins. This study compared the properties of cells from the myocardial sleeves of the pulmonary veins (PV(m)) with cells from the normal cardiac pacemaker (the sinoatrial node) and regions of the atria. Our objective was to identify key features of these cells that predispose them to becoming the focus of cardiac arrhythmias. METHODS AND RESULTS: Single cells were isolated from samples of rabbit PV(m), central and peripheral sinoatrial node, crista terminalis, and left and right atria. Detailed morphology of cells was assessed and intracellular calcium concentrations measured with the use of Fluo-3. Cells from the PV(m) were smaller than atrial cells and showed large elevations in diastolic calcium during activation at physiological rates, a feature the PV(m) cells shared with cells from the sinoatrial node. Unstimulated spontaneous activity was observed in a minority of cells from the PV(m), but numerous cells from this region showed spontaneous activity for a brief period immediately subsequent to stimulation at physiological rates. This was not observed in atrial cells. Assessment of calcium removal pathways showed sarcolemmal calcium extrusion in cells from the PV(m) to have a high reliance on "slow" extrusion pathways to maintain intracellular calcium homeostasis because of a low expression of sodium-calcium exchanger. CONCLUSIONS: We conclude that cells from the PV(m) share some features with cells from the sinoatrial node but also have distinctly unique features that predispose them to the development of spontaneous activity.

Declining into failure: the age-dependent loss of the L-type calcium channel within the sinoatrial node.

BACKGROUND: The spontaneous activity of pacemaker cells in the sinoatrial (SA) node controls heart rate under normal physiological conditions. Clinical studies have shown the incidence of SA node dysfunction increases with age and occurs with peak prevalence in the elderly population. The present study investigated whether aging affected the expression of Ca(v)1.2 channels and whether these changes could affect pacemaker activity, in turn leading to age-related SA node degeneration. METHODS AND RESULTS: The SA node region was isolated from the right atrium of guinea pigs between birth and 38 months of age. Immunofluorescence studies showed Ca(v)1.2 protein was present as punctate labeling around the outer membrane of atrial cells but was absent from the center of the SA node. The area lacking Ca(v)1.2-labeled protein progressively increased from 2.06+/-0.1 (mean+/-SEM) mm2 at 1 month to 18.72+/-2.2 mm2 at 38 months (P<0.001). Western blot provided verification that Ca(v)1.2 protein expression within the SA node declined during aging. Functional measurements showed an increased sensitivity to the L-type calcium blocker nifedipine; SA node preparations stopped beating in 100 micromol/L nifedipine at 1 day old, compared with 30 micromol/L at 1 month and 10 micromol/L at 38 months of age. Furthermore, the amplitude of extracellular potentials declined within the center and periphery of the SA node during aging. CONCLUSIONS: The present data show Ca(v)1.2 channel protein decreases concurrently with reduced spontaneous activity of the SA node with increased age, which provides further evidence of mechanisms underlying the age-related deterioration of the cardiac pacemaker.

Ageing-related changes of connexins and conduction within the sinoatrial node.

Clinical studies have shown that sinoatrial node dysfunction occurs at the highest incidence in the elderly population. Guinea-pigs were studied throughout their lifespan (i.e. birth to 38 months) to investigate the possible mechanism leading to nodal dysfunction. Using immunofluorescence with confocal microscopy, Cx43 protein expression was shown at birth to be present throughout the sinoatrial node and atrial muscle, however, at one month Cx43 protein was not expressed in the centre of the sinoatrial node. Throughout the remainder of the animal's lifespan the area of tissue lacking Cx43 protein progressively increased. Western blot provided verification by quantitative analysis that Cx43 protein expression within the sinoatrial node decreased with age; however, the expression of other cardiac connexins, Cx40 and Cx45, did not differ with age. Analysis of conduction maps showing propagation of the action potential across the sinoatrial node, from the initiation point to the crista terminalis, found that the action potential conduction time taken and conduction distance increased proportionally with age; conversely the conduction velocity decreased with age. We have shown ageing induces degenerative changes in action potential conduction, contributed to by the observed loss of Cx43 protein. Our data identify Cx43 as a potential therapeutic target for quashing the age-related deterioration of the cardiac pacemaker.

Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking.

The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2-6 microm) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (KD, 1-10 nm) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na+ currents under physiological conditions and the expression of cardiac and neuronal Na+ channel isoforms. We identified two distinct Na+ current components, TTX resistant and TTX sensitive. At 37 degrees C, TTX-resistant iNa and TTX-sensitive iNa started to activate at approximately -70 and approximately -60 mV, and peaked at -30 and -10 mV, with a current density of 22 +/- 3 and 18 +/- 1 pA pF(-1), respectively. TTX-sensitive iNa inactivated at more positive potentials as compared to TTX-resistant iNa. Using action potential clamp, TTX-sensitive iNa was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Nav1.5, and the TTX-sensitive neuronal isoform, Nav1.1, were observed: Nav1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Nav1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nm) of TTX, which block TTX-sensitive iNa, slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 microm) of TTX, which block TTX-resistant iNa as well as TTX-sensitive iNa, slowed both pacemaking and SA node conduction. It is concluded that two Na+ channel isoforms are important for the functioning of the SA node: neuronal (putative Nav1.1) and cardiac Nav1.5 isoforms are involved in pacemaking, although the cardiac Nav1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.

Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control.

Recent studies have proposed that release of calcium from the sarcoplasmic reticulum (SR) modulates the spontaneous activity of the sinoatrial node (SAN). Previously we have shown that several calcium regulatory proteins are expressed at a lower level in the centre of the SAN compared with the periphery. Such differences may produce heterogeneity of intracellular calcium handling and pacemaker activity across the SAN. Selective isolations showed that the centre of the SAN is composed of smaller cells than the periphery. Measurements of cytosolic calcium in spontaneously beating cells showed that diastolic calcium, systolic calcium, the calcium transient amplitude and spontaneous rate were greater in larger (likely to be peripheral) cells compared with smaller (likely to be central) SAN cells. The SR calcium content was greater in larger cells, although SR recruitment was more efficient in smaller cells. The sodium-calcium exchanger and sarcolemmal calcium ATPase had a lower activity and the exchanger was responsible for a larger proportion of sarcolemmal calcium extrusion in smaller cells compared with larger cells. Ryanodine had a greater effect on the spontaneous calcium transient in larger cells compared with smaller cells, and slowed pacemaker activity in larger cells but not smaller cells, thus abolishing the difference in cycle length. This study shows heterogeneity of intracellular calcium regulation within the SAN and this contributes to differences in pacemaker activity between cells from across the SAN. The smallest central cells of the leading pacemaker region of the SAN do not require SR calcium for spontaneous activity nor does disruption of the SR alter pacemaking in these primary pacemaker cells.

Sophisticated architecture is required for the sinoatrial node to perform its normal pacemaker function.

The heart's pacemaker, the sinoatrial node, does not consist of a group of uniform sinoatrial node cells embedded in atrial muscle. Instead, it is a heterogeneous tissue with multiple cell types and a complex structure. Evidence suggests that from the periphery to the center of the sinoatrial node, there is a gradient in action potential shape, pacemaking, ionic current densities, connexin expression, Ca2+ handling, myofilament density, and cell size. This complexity may be necessary for the sinoatrial node to pacemake under diverse conditions, drive the more hyperpolarized atrial muscle, and resist proarrhythmic perturbations.

Cx43 and dual-pathway electrophysiology of the atrioventricular node and atrioventricular nodal reentry.

Fluorescent imaging has revealed that posterior nodal extensions provide the anatomical substrate for the dual-pathway electrophysiology of the atrioventricular (AV) node during normal conduction and reentry. The reentry can be intranodal, or as well as the posterior nodal extensions, it can involve an endocardial layer of atrial/atrial-nodal (A/AN) cells as part of the AV nodal reentry (AVNR) circuit. Using fluorescent imaging with a voltage-sensitive dye and immunolabeling of Cx43, we mapped the electrical activity and structural substrate in 3 types of AVNR induced by premature atrial stimulation in 8 rabbit hearts. In 6 cases, the AVNR pathway involved (1) a fast pathway (FP), (2) the A/AN layer, and (3) a slow pathway (SP). In 4 cases, reentry took the path (1) SP, (2) A/AN layer, and (3) FP. In 2 cases, reentry was intranodal, propagating between the 2 posterior nodal extensions. Immunolabeling revealed that the FP and SP are formed by Cx43-expressing bundles surrounded by tissue without Cx43. Cx43-expressing posterior nodal extensions are the substrate of AVNR during both intranodal and extranodal reentry.