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Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2DeltaCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin's effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin.

The prevention and treatment of cardiac arrhythmias conferring major morbidity and mortality is far from optimal, and relies heavily on devices and drugs for the partial successes that have been seen. The greatest success has been in the use of electronic pacemakers to drive the hearts of patients having high degree heart block. Recent years have seen the beginnings of attempts to use novel approaches available through gene and cell therapies to treat both brady- and tachyarrhythmias. By far the most successful approaches to date have been seen in the development of biological pacemakers. However, the far more difficult problems posed by atrial fibrillation and ventricular tachycardia are now being addressed. In the following pages we review the approaches now in progress as well as the specific methodologic demands that must be met if these therapies are to be successful.

Recent results indicate that phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), directly enhance the opening of hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels by shifting their activation gating to more positive voltages. This contrasts with the action of phosphoinositides to inhibit the opening of the related cyclic nucleotide-gated (CNG) channels involved in sensory signaling. We both review previous studies and present new experiments that investigate whether HCN channels may be regulated by dynamic changes in PI(4,5)P(2) levels caused by the receptor-mediated activation of phospholipase C (PLC). We coexpressed HCN1 or HCN2 channels in Xenopus oocytes with the PLC-coupled bradykinin BK(2) receptor, the muscarinic M1 receptor, or the TrkA receptor. Activation of all three receptors produced a positive shift in HCN channel voltage gating, the opposite of the effect expected for PI(4,5)P(2) depletion. This action was not caused by alterations in cAMP as the effect was preserved in HCN mutant channels that fail to bind cAMP. The receptor effects were mediated by PLC activity, but did not depend on signaling through the downstream products of PI(4,5)P(2) hydrolysis: IP(3) or diacylglycerol (DAG). Importantly, the modulatory effects on gating were blocked by inhibitors of phosphatidylinositol (PI) kinases, suggesting a role for increased PI(4,5)P(2) synthesis. Finally, we found that bradykinin exerted a similar PI kinase-dependent effect on the gating of native HCN channels in cardiac sinoatrial node cells, suggesting that this pathway may represent a novel, physiologically relevant mechanism for enhancing HCN channel function.

Biological pacemaking as a replacement for or adjunct to electronic pacemakers has been a subject of interest since the 1990s. In the following pages, we discuss the rational for and progress made using a hyperpolarization activated, cyclic nucleotide gated channel isoform to carry the I(f) pacemaker current in gene and cell therapy approaches. Both strategies have resulted in effective biological pacemaker function over a period of weeks in intact animals. Moreover, the use of adult human mesenchymal stem cells as a platform for carrying pacemaker genes has resulted in the formation of functional gap junctions with cardiac myocytes in situ leading to effective and persistent propagation of pacemaker current. The approaches described are encouraging, suggesting that biological pacemakers based on this strategy can be brought to clinical testing.

Pacemaker current (I(f)) encoded by the HCN gene family contributes importantly to cardiac rhythm. That contribution depends on the biophysical characteristics of I(f), such as voltage dependence, which vary markedly with cardiac region, development and disease. Heterologous expression studies of individual HCN isoforms have failed to account for the diverse functionality of the native current. To investigate the influence of cellular environment on the gating of HCN channels, we compared the functional characteristics of HCN2 and HCN4, the two major ventricular isoforms, when over-expressed in a normal context (neonatal myocytes) and in a heterologous context (HEK 293 cells). Independent of cell type, HCN4 activates substantially slower than HCN2 and with a half-maximum activation voltage approximately equal 10 mV less negative. However, both isoforms activate more positively in myocytes than in HEK 293 cells. The latter result suggests a context dependence (i.e. cell-type specificity) to HCN voltage dependence that exerts a comparable influence on these two isoforms. This is distinct from the inherent difference in the biophysical properties of HCN2 and HCN4.

We have reported previously that the sinoatrial node (SAN) in the newborn rabbit expresses a Na+ current (INa) with properties similar to the neuronal type-I isoform and that this current contributes to the net inward current flowing during diastolic depolarization. To characterize this current further we conducted cell-attached single-channel experiments in isolated newborn SAN myocytes. The Na+ channel was sensitive to divalent cation block and had a single-channel conductance of 25.6 pS in the absence of divalent cations. Kinetic compatibility between single-channel and previous whole-cell data was confirmed by measuring the time constant of current decay. At pacemaker potentials, time constants were of the order of tens of milliseconds. Additional experiments indicated that this slow inactivation arises because the Na+ channels expressed in the neonatal SAN tend to re-open frequently at potentials in the pacemaker range. We suggest that this is the mechanism by which a small tetrodotoxin (TTX)-sensitive current contributes to the total inward current flowing during slow diastolic depolarization in neonatal (but not adult) pacemaker myocytes.

AIDS dementia complex affects 15-20% of HIV-infected adults and a greater percentage of HIV-infected children. Whether or not an HIV-infected individual develops neurological disease and how early in infection the clinical signs appear is most likely the net result of both viral virulence factors and host factors. Important viral factors include cell tropism and sequences that determine neurovirulence. The host factors include the cellular expression of viral co-receptors and maintenance of competent immune responses. The pathogenesis of AIDS dementia complex is difficult to study in the human host because of the difficulty in identifying acutely infected individuals and the inaccessibility of human brain tissue for examination during infection. The SIV/macaque model is excellent for the study of viral virulence factors and host responses to infection. This review outlines how the SIV/macaque model has been used to identify viral factors that are important for the development of neurological disease, to determine when HIV enters the brain, and to characterize the host immune responses affecting virus entry to the CNS and the development of neurological disease.

Myocytes were isolated from newborn and adult rat ventricle. Using the whole-cell patch clamp, the two cell populations were compared for the presence of the hyperpolarization-activated pacemaker current if. As in other mammalian species, the threshold voltage in acutely dissociated adult rat myocytes was extremely negative (-113 +/- 5 mV; n=12). In contrast, threshold in newborn cells was relatively positive, regardless of whether measured in acutely dissociated (-72 +/- 2 mV; n=6) or cultured cells (-70 +/- 2 mV; n=9). Current density was not reduced in the adult. These results suggest that with development the ventricle assumes its non-pacemaker function, at least in part, by a shift of the voltage dependence of if outside the physiological range.

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to determine whether their actions can be related to the overall immunological rejection of the transplanted heart. Granule-induced myocyte morphological changes and final destruction were preceded by shortening of action potential duration (APD) and reductions of the resting potential and the action potential amplitude. APD shortening was probably caused by a granule-induced increase in outward current (most likely non-specific). Ryanodine, which blocks Ca2+ release from the sarcoplasmic reticulum, did not interfere with the morphological and electrophysiological effects of lytic granules. Fura-2 imaging indicated that [Ca2+]i initially increased about 2-fold from 90.0 +/- 11.5 nM, while cell length decreased less than 5% from a mean value of 99.0 +/- 9.0 microns. A further increase in [Ca2+]i (greater than 10 fold) was associated with progressive contracture and destruction, suggesting that the structural damage inflicted by lytic granules is caused by [Ca2+]i overload. The results indicate that the cytocidal action of CTL-derived lytic granules may be involved in immunologically induced damage, even to the extent of rejection of the transplanted heart.

Ion sensitive indicator molecules can be employed in conjunction with fluorescence microscopy in single cells to measure rapid changes in the intracellular concentration of several ionic species. A number of these probes (e.g. fura-2) require the capability of measuring emission intensity at two excitation wavelengths to quantitate properly intracellular ion concentration. We have developed a simple dual-excitation apparatus for use in such applications. The apparatus switches the excitation filter within 150 ms. This economical apparatus is well suited in situations where the ionic concentration of interest is changing relatively slowly. Moreover, by synchronizing the device's action with an external stimulus, rapid and reproducible ionic changes in excitable tissue also can be measured.

Guanine nucleotide binding proteins were examined for their influence in developmental and adaptive models of adrenergic actions in the heart. In primary cultures of rat cardiac myocytes, the positive chronotropic response to the alpha-agonist, phenylephrine, changes to negative when these cells are grown with and innervated by sympathetic nerves from the paravertebral chain. Innervated cells have significantly more G protein, as determined by the ADP-ribosylation reaction catalyzed by pertussis toxin, which is linked functionally to the negative chronotropic response. Adult canine Purkinje fibers that respond to phenylephrine with a decrease in automaticity are also linked biochemically and functionally to a G protein that serves as a pertussis toxin substrate. Fibers that increase in automaticity after exposure to phenylephrine, either under control conditions (a minority of fibers) or after prior exposure to pertussis toxin (a majority of fibers), have markedly reduced levels of G. A G protein was also shown to be important in the blunted adrenergic responsiveness that characterizes congestive heart failure in human subjects. In this model, the receptor complex is beta-adrenergic and the involved G protein is a cholera toxin substrate. Gs is reduced in the lymphocytes of patients with congestive heart failure and increases toward normal after successful therapy. These observations highlight the important roles that G proteins have in adrenergic actions of the heart both with respect developmental and adaptive changes.