Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes.

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca2+ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, If, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (-70 to -50 mV), which corresponds to the 'pacemaker depolarization' in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of If channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in If, and the well-known increases in the L-type Ca2+ current, ICaL, demonstrated that ICaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, ICaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing If, our simulations reveal a novel functional component of If in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of If and opening of ICaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.

Monocyte chemotactic protein-1 inhibits chondrogenesis of synovial mesenchymal progenitor cells: an in vitro study.

Osteoarthritis (OA) is a multifactorial, often progressive, painful disease. OA often progresses with an apparent irreversible loss of articular cartilage, exposing underlying bone, resulting in pain and loss of mobility. This cartilage loss is thought to be permanent due to ineffective repair and apparent lack of stem/progenitor cells in that tissue. However, the adjacent synovial lining and synovial fluid are abundant with mesenchymal progenitor/stem cells (synovial mesenchymal progenitor cells [sMPCs]) capable of differentiating into cartilage both in vitro and in vivo. Previous studies have demonstrated that MPCs can home to factors such as monocyte chemotactic protein 1 (MCP-1/CCL2) expressed after injury. While MCP-1 (and its corresponding receptors) appears to play a role in recruiting stem cells to the site of injury, in this study, we have demonstrated that MCP-1 is upregulated in OA synovial fluid and that exposure to MCP-1 activates sMPCs, while concurrently inhibiting these cells from undergoing chondrogenesis in vitro. Furthermore, exposure to physiological (OA knee joint synovial fluid) levels of MCP-1 triggers changes in the transcriptome of sMPCs and prolonged exposure to the chemokine induces the expression of MCP-1 in sMPCs, resulting in a positive feedback loop from which sMPCs cannot apparently escape. Therefore, we propose a model where MCP-1 (normally expressed after joint injury) recruits sMPCs to the area of injury, but concurrently triggers changes in sMPC transcriptional regulation, leading to a blockage in the chondrogenic program. These results may open up new avenues of research into the lack of endogenous repair observed after articular cartilage injury and/or arthritis.

Two-pore domain K⁺ channels regulate membrane potential of isolated human articular chondrocytes.

Potassium channels that regulate resting membrane potential (RMP) of human articular chondrocytes (HACs) of the tibial joint maintained in short-term (0-3 days) non-confluent cell culture were studied using patch-clamp techniques. Quantitative PCR showed that transcripts of genes for two-pore domain K(+) channels (KCNK1, KCNK5 and KCNK6), and 'BK' Ca(2+)-activated K(+) channels (KCNMA1) were abundantly expressed. Immunocytological methods detected α-subunits for BK and K(2p)5.1 (TASK-2) K(+) channels. Electrophysiological recordings identified three distinct K(+) currents in isolated HACs: (i) a voltage- and time-dependent 'delayed rectifier', blocked by 100 nM α-dendrotoxin, (ii) a large 'noisy' voltage-dependent current that was blocked by low concentrations of tetraethylammonium (TEA; 50% blocking dose = 0.15 mM) and iberiotoxin (52% block, 100 nM) and (iii) a voltage-independent 'background' K(+) current that was blocked by acidic pH (5.5-6), was increased by alkaline pH (8.5), and was not blocked by TEA, but was blocked by the local anaesthetic bupivacaine (0.25 mM). The RMP of isolated HACs was very slightly affected by 5 mM TEA, which was sufficient to block both voltage-dependent K(+) currents, suggesting that these currents probably contributed little to maintaining RMP under 'resting' conditions (i.e. low internal [Ca(2+)]). Increases in external K(+) concentration depolarized HACs by 30 mV in response to a 10-fold increase in [K(+)], indicating a significant but not exclusive role for K(+) current in determining RMP. Increases in external [K(+)] in voltage-clamped HACs revealed a voltage-independent K(+) current whose inward current magnitude increased with external [K(+)]. Block of this current by bupivacaine (0.25-1 mM) in 5 and 25 mM external [K(+)] resulted in a large (8-25 mV) depolarization of RMP. The biophysical and pharmacological properties of the background K(+) current, together with expression of mRNA and α-subunit protein for TASK-2, strongly suggest that these two-pore domain K(+) channels contribute significantly to stabilizing the RMP of HACs.

Dilated cardiomyopathy is associated with reduced expression of the cardiac sodium channel Scn5a.

OBJECTIVE: Dilated cardiomyopathy (DCM) leads to dilation of the cardiac chambers and congestive heart failure. Recent reports have associated mutations in the SCN5A gene, which codes for the major cardiac sodium channel Nav1.5, with DCM. Although DCM is the most common form of cardiomyopathy, no animal studies have established this functional connection. METHODS AND RESULTS: We have produced transgenic mice that ectopically express the transcriptional repressor Snail in heart. These animals display severe DCM, ECG abnormalities, conduction defects, revealed by voltage-sensitive dye imaging, and significantly reduced voltage-gated sodium current as measured by patch clamping. There is a concomitant decrease in expression of the major cardiac sodium channel gene Scn5a, which we show by gene reporter assays and electrophoretic mobility shift assays is a direct target of Snail. CONCLUSIONS: Our findings indicate that a decrease in Scn5a expression and significant reduction in sodium current can result in DCM, and support the hypothesis that some mutations in the human SCN5A gene can lead to DCM.

Na(+) current expression in human atrial myofibroblasts: identity and functional roles.

In the mammalian heart fibroblasts have important functional roles in both healthy conditions and diseased states. During pathophysiological challenges, a closely related myofibroblast cell population emerges, and can have distinct, significant roles. Recently, it has been reported that human atrial myofibroblasts can express a Na(+) current, INa. Some of the biophysical properties and molecular features suggest that this INa is due to expression of Nav 1.5, the same Na(+) channel α subunit that generates the predominant INa in myocytes from adult mammalian heart. In principle, expression of Nav 1.5 could give rise to regenerative action potentials in the fibroblasts/myofibroblasts. This would suggest an active as opposed to passive role for fibroblasts/myofibroblasts in both the "trigger" and the "substrate" components of cardiac rhythm disturbances. Our goals in this preliminary study were: (i) to confirm and extend the electrophysiological characterization of INa in a human atrial fibroblast/myofibroblast cell population maintained in conventional 2-D tissue culture; (ii) to identify key molecular properties of the α and ß subunits of these Na(+) channel(s); (iii) to define the biophysical and pharmacological properties of this INa; (iv) to integrate the available multi-disciplinary data, and attempt to illustrate its functional consequences, using a mathematical model in which the human atrial myocyte is coupled via connexins to fixed numbers of fibroblasts/myofibroblasts in a syncytial arrangement. Our experimental findings confirm that a significant fraction (approximately 40-50%) of these human atrial myofibroblasts can express INa. However, our data suggest that INa may be generated by a combination of Nav 1.9, Nav 1.2, and Nav 1.5. Our results, when complemented with mathematical modeling, provide a background for re-evaluating pharmacological management of supraventricular rhythm disorders, e.g., persistent atrial fibrillation.

Voltage-gated K+ currents in mouse articular chondrocytes regulate membrane potential.

Membrane currents and resting potential of isolated primary mouse articular chondrocytes maintained in monolayer cell culture for 1-9 days were recorded using patch clamp methods. Quantitative RT-PCR showed that the most abundantly expressed transcript of voltage-gated K(+) channels was for K(V)1.6, and immunological methods confirmed the expression of K(V)1.6 α-subunit proteins. These chondrocytes expressed a large time- and potential-dependent, Ca(2+)-independent 'delayed rectifier' K(+) current. Steady-state activation was well-fit by a Boltzmann function with a threshold near -50 mV, and a half-activation potential of -34.5 mV. The current was 50% blocked by 1.48 mM tetraethylammonium, 0.66 mM 4-aminopyridine and 20.6 nM α-dendrotoxin. The current inactivated very slowly at membrane potentials in the range of the resting potential of the chondrocytes. Resting membrane potential of the chondrocytes at room temperature (19-21°C) and in 5 mM external K(+) was -46.4 ± 1.3 mV (mean ± s.e.m; n = 23), near the 'foot' of the activation curve of this K(+) current. Resting potential was depolarized by an average of 4.2 ± 0.8 mV by 25 mM TEA, which blocked about 95% of the K(+) current. At a membrane potential of -50 mV, the apparent time constant of inactivation (tau(in)) was 37.9 s, and the 'steady-state' current level was 19% of that at a holding potential of -90 mV; at -40 mV, tau(in) was 20.3 s, and 'steady-state' current was 5% of that at -90 mV. These results demonstrate that in these primary cultured, mouse articular chondrocytes steady-state activation of a voltage-gated K(+) current contributes to resting membrane potential. However, this current is also likely to have a significant physiological role in repolarizing the chondrocyte following depolarizing stimuli that might occur in conditions of membrane stretch. For example, activation of TRP('transient receptor potential') non-specific cation channels in these cells during cyclic loading and unloading of the joint cartilage, or in response to hypertonic challenge is expected to result in depolarization and Ca(2+) entry. Potassium currents are required to maintain the resting membrane potential.

Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes.

Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K(+) current (I(K(ACh))) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by approximately 25% (vs. untreated controls) in field-stimulated myocytes. S1P(1) was shown to be involved by using the S1P(1)-selective agonist SEW2871 on myocytes isolated from S1P(3)-null mice. However, in these myocytes, S1P(3) can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P(1) antagonist VPC23019. Since S1P(1) activates G(i) exclusively, whereas S1P(3) activates both G(i) and G(q), these results strongly implicate the involvement of mainly G(i). Additional experiments using the I(K(ACh)) blocker tertiapin demonstrated that I(K(ACh)) can contribute to the negative inotropy following S1P activation of S1P(1) (perhaps through G(ibetagamma) subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I(K(ACh))) can reduce L-type calcium current and thus can decrease the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G(i) to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca(2+)](i); and 2) a second pathway that acts via G(i) to activate I(K(ACh)) and reduce APD. This decrease in APD is expected to decrease Ca(2+) influx and reduce [Ca(2+)](i) and myocyte contractility.

Comparison of time- and voltage-dependent K+ currents in myocytes from left and right atria of adult mice.

Consistent differences in K+ currents in left and right atria of adult mouse hearts have been identified by the application of current- and voltage-clamp protocols to isolated single myocytes. Left atrial myocytes had a significantly (P < 0.05) larger peak outward K+ current density than myocytes from the right atrium. Detailed analysis revealed that this difference was due to the rapidly activating sustained K+ current, which is inhibited by 100 muM 4-aminopyridine (4-AP); this current was almost three times larger in the left atrium than in the right atrium. Accordingly, 100 muM 4-AP caused a significantly (P < 0.05) larger increase in action potential duration in left than in right atrial myocytes. Inward rectifier K+ current density was also significantly (P < 0.05) larger in left atrial myocytes. There was no difference in the voltage-dependent L-type Ca2+ current between left and right atria. As expected from this voltage-clamp data, the duration of action potentials recorded from single myocytes was significantly (P < 0.05) shorter in myocytes from left atria, and left atrial tissue was found to have a significantly (P < 0.05) shorter effective refractory period than right atrial tissue. These results reveal similarities between mice and other mammalian species where the left atrium repolarizes more quickly than the right, and provide new insight into cellular electrophysiological mechanisms responsible for this difference. These findings, and previous results, suggest that the atria of adult mice may be a suitable model for detailed studies of atrial electrophysiology and pharmacology under control conditions and in the context of induced atrial rhythm disturbances.

Effects of C-type natriuretic peptide on ionic currents in mouse sinoatrial node: a role for the NPR-C receptor.

The effects of C-type natriuretic peptide (CNP) on heart rate and ionic currents were demonstrated by recording the ECG from adult mice and performing voltage-clamp experiments on single sinoatrial (SA) node cells isolated from mouse heart. The selective natriuretic peptide type C receptor (NPR-C) agonist cANF (10(-7) M) significantly decreased heart rate in the presence of isoproterenol (5 x 10(-9) M), as indicated by an increase in the R-R interval of ECGs obtained from Langendorff-perfused hearts. Voltage-clamp measurements in enzymatically isolated single pacemaker myocytes revealed that CNP (10(-8) M) and cANF (10(-8) M) significantly inhibited L-type Ca2+ current [ICa(L)]. These findings suggest that the CNP effect on this current is mediated by NPR-C. Further support for an NPR-C-mediated inhibition of ICa(L) in SA node myocytes was obtained by altering the functional coupling between the G protein Gi and NPR-C. In these experiments, a "Gi-activator peptide," which consists of a 17-amino acid segment of NPR-C containing a specific Gi protein-activator sequence, was dialyzed into SA node myocytes. This peptide decreased ICa(L) significantly, suggesting that NPR-C activation can result in a reduction in ICa(L) when CNP is bound and the Gi protein pathway is activated. This effect of CNP appears to be selective for ICa(L), because the hyperpolarization-activated current was unaffected by CNP or cANF. These results provide the first demonstration that CNP has a negative chronotropic effect on heart rate and suggest that this effect is mediated by selectively activating NPR-C and reducing ICa(L) through coupling to Gi protein.