Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

C-terminal domain of Kv4.2 and associated KChIP2 interactions regulate functional expression and gating of Kv4.2

The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.     

Multi-Walled Carbon Nanotubes Impair Kv4.2/4.3 Channel Activities,Delay Membrane Repolarization and Induce Bradyarrhythmias in the Rat

Purpose

The potential hazardous effects of multi-walled carbon nanotubes (MWCNTs) on cardiac electrophysiology are seldom evaluated. This study aimed to investigate the impacts of MWCNTs on the Kv4/I _to channel, action potential and heart rhythm and the underlying mechanisms.

Methods

HEK293 cells were engineered to express Kv4.2 or Kv4.3 with or without KChIP2 expression. A series of approaches were introduced to analyze the effects of MWCNTs on Kv4/I _to channel kinetics, current densities, expression and trafficking. Transmission electron microscopy was performed to observe the internalization of MWCNTs in HEK293 cells and rat cardiomyocytes. Current clamp was employed to record the action potentials of isolated rat cardiomyocytes. Surface ECG and epicardial monophasic action potentials were recorded to monitor heart rhythm in rats in vivo. Vagal nerve discharge monitoring and H&E staining were also performed.

Results

Induction of MWCNTs into the cytosole through pipette solution soon accelerated the decay of I _Kv4 in HEK293 cells expressing Kv4.2/4.3 and KChIP2, and promoted the recovery from inactivation when Kv4.2 or Kv4.3 was expressed alone. Longer exposure (6 h) to MWCNTs decreased the I _Kv4.2 density, Kv4.2/Kv4.3 (but not KChIP2) expression and trafficking towards the plasma membrane in HEK293 cells. In acutely isolated rat ventricular myocytes, pipette MWCNTs also quickly accelerated the decay of I _Kv4 and prolonged the action potential duration (APD). Intravenous infusion of MWCNTs (2 mg/rat) induced atrioventricular (AV) block and even cardiac asystole. No tachyarrhythmia was observed after MWCNTs administration. MWCNTs did not cause coronary clot but induced myocardial inflammation and increased vagus discharge.

Conclusions

MWCNTs suppress Kv4/I _to channel activities likely at the intracellular side of plasma membrane, delay membrane repolarization and induce bradyarrhythmia. The delayed repolarization, increased vagus output and focal myocardial inflammation may partially underlie the occurrence of bradyarrhythmias induced by MWCNTs. The study warns that MWCNTs are hazardous to cardiac electrophysiology.     

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable,Depending on the Relative Expression Level

Kv4 is a voltage-gated K⁺ channel, which underlies somatodendritic subthreshold A-type current (I_SA) and cardiac transient outward K⁺ (I_to) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K⁺ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.     

Selegiline improves cardiac sympathetic terminal function and beta-adrenergic responsiveness in heart failure

Selegiline is a centrally acting sympatholytic agent with neuroprotective properties. It also has been shown to promote sympathetic reinnervation after sympathectomy. These actions of selegiline may be beneficial in heart failure that is characterized by increased sympathetic nervous activity and functional sympathetic denervation. Twenty-seven rabbits with rapid cardiac pacing (360 beats/min, 8 wk) and twenty-three rabbits without pacing were randomly assigned to receive selegiline (1 mg/day, 8 wk) or placebo. Rapid pacing increased plasma norepinephrine (NE) and decreased left ventricular fractional shortening, baroreflex sensitivity, cardiac sympathetic nerve terminal profiles, cardiac NE uptake activity, and myocardial beta-adrenoceptor density. Selegiline administration to animals with rapid ventricular pacing attenuated the increase in plasma NE and decreases in fractional shortening, baroreflex sensitivity, sympathetic nerve profiles, NE uptake activity and beta-adrenoceptor density. Thus selegiline appears to exert a sympatholytic and cardiac neuroprotective effect in pacing-induced cardiomyopathy. The effects are potentially beneficial because selegiline not only improves cardiac function but also increases baroreflex sensitivity in heart failure.     

Interaction of syntaxin 1A with the N-terminus of Kv4.2 modulates channel surface expression and gating

Kv4.2 channels are responsible in the heart for the Ca2+-independent transient outward currents and are important in regulating myocardial excitability and Ca2+ homeostasis. We have identified previously the expression of syntaxin 1A (STX1A) on the cardiac ventricular myocyte plasma membranes, and its modulation of cardiac ATP-sensitive K+ channels. We speculated that STX1A interacts with other cardiac ion channels, thus we examined the interaction of STX1A with Kv4.2 channels. Co-immunoprecipitation and GST pulldown assays demonstrated a direct interaction of STX1A with the Kv4.2 N-terminus. We next investigated the functional alterations of Kv4.2 gating by STX1A in Xenopus oocytes. Coexpression of Kv4.2 with STX1A (1) resulted in a reduction of Kv4.2 current amplitude; (2) caused a depolarizing shift of the steady-state inactivation curve; (3) enhanced the rate of current decay; and (4) accelerated the rate of recovery from inactivation. Additional coexpression of botulinum neurotoxin C, which cleaves STX1A, reversed the effects of STX1A on Kv4.2. STX1A inhibited partially the gating changes by KChIP2, suggesting a competitive interaction of these proteins for an overlapping binding region on the N-terminus of Kv4.2. Indeed, the N-terminal truncation mutants of Kv4.2 (Kv4.2Delta2-40 and Kv4.2Delta7-11), which form part of the KChIP2 binding site, displayed reduced sensitivity to STX1A modulation. Our study suggests that STX1A directly modulates Kv4.2 current amplitude and gating through its interaction with an overlapping region of the KChIP binding motif domain on the Kv4.2 N-terminus.     

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five- fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage- gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.     

A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia.

KChIP2, a gene encoding three auxiliary subunits of Kv4.2 and Kv4.3, is preferentially expressed in the adult heart, and its expression is downregulated in cardiac hypertrophy. Mice deficient for KChIP2 exhibit normal cardiac structure and function but display a prolonged elevation in the ST segment on the electrocardiogram. The KChIP2(-/-) mice are highly susceptible to the induction of cardiac arrhythmias. Single-cell analysis revealed a substrate for arrhythmogenesis, including a complete absence of transient outward potassium current, I(to), and a marked increase in action potential duration. These studies demonstrate that a defect in KChIP2 is sufficient to confer a marked genetic susceptibility to arrhythmias, establishing a novel genetic pathway for ventricular tachycardia via a loss of the transmural gradient of I(to).     

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2.

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels.     

Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes

The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base.     

Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy

Regulation of voltage-gated K(+) (K(v)) channel expression may be involved in controlling contractility of uterine smooth muscle cells during pregnancy. Functional expression of these channels is not only controlled by the levels of pore-forming subunits, but requires their association with auxiliary subunits. Specifically, rapidly inactivating K(v) current is prominent in myometrial cells and may be carried by complexes consisting of Kv4 pore-forming and KChIP auxiliary subunits. To determine the molecular identity of the channel complexes and their changes during pregnancy, we examined the expression and localization of these subunits in rat uterus. RT-PCR analysis revealed that rat uterus expressed all three Kv4 pore-forming subunits and KChIP2 and -4 auxiliary subunits. The expression of mRNAs for these subunits was dynamically and region selectively regulated during pregnancy. In the corpus, Kv4.2 mRNA level increased before parturition, whereas the expression of Kv4.1 and Kv4.3 mRNAs decreased during pregnancy. A marked increase in KChIP2 mRNA level was also seen at late gestation. In the cervix, the expression of all three pore-forming and two auxiliary subunit mRNAs increased at late gestation. Immunoprecipitation followed by immunoblot analysis indicated that Kv4.2-KChIP2 complexes were significant in uterus at late pregnancy. Kv4.2- and KChIP2-immunoreactive proteins were present in both circular and longitudinal myometrial cells. Finally, Kv4.2 and KChIP2 mRNA levels were similarly elevated in pregnant and nonpregnant corpora of one side-conceived rats. These results suggest that diffusible factors coordinate the pregnancy-associated changes in molecular compositions of myometrial Kv4-KChIP channel complexes.     

Augmentation of Kv4.2-encoded currents by accessory dipeptidyl peptidase 6 and 10 subunits reflects selective cell surface Kv4.2 protein stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.     

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.     

Functional stoichiometry underlying KChIP regulation of Kv4.2 functional expression

K channel‐interacting proteins (KChIPs) enhance functional expression of Kv4 channels by binding to an N‐terminal regulatory region located in the first 40 amino acids of Kv4.2 that we call the functional expression regulating N‐terminal (FERN) domain. Mutating two residues in the FERN domain to alanines, W8A and F11A, disrupts KChIP binding and regulation of Kv4.2 without eliminating the FERN domain's control of basal expression level or regulation by DPP6. When Kv4.2(W8A,F11A) is co‐expressed with wild type Kv4.2 and KChIP3 subunits, a dominant negative effect is seen where the current expression is reduced to levels normally seen without KChIP addition. The dominant negative effect correlates with heteromultimeric channels remaining on intracellular membranes despite KChIP binding to non‐mutant Kv4.2 subunits. In contrast, the deletion mutant Kv4.2(Δ1‐40), eliminating both KChIP binding and the FERN domain, has no dominant negative effect even though the maximal conductance level is 5x lower than seen with KChIP3. The 5x increased expression seen with KChIP integration into the channel is fully apparent even when a reduced number of KChIP subunits are incorporated as long as all FERN domains are bound. Our results support the hypothesis that KChIPs enhances Kv4.2 functional expression by a 1 : 1 suppression of the N‐terminal FERN domain and by producing additional positive regulatory effects on functional channel expression.     

A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels

Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 alpha subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 alpha subunits. Co-expression of KChIPs1-3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice.     

Rat heart GDNF: effect of chemical sympathectomy

Developmental studies indicate a role for GDNF in survival of motor, autonomic, and sensory neurons. However, no study attempted to demonstrate its participation in autonomic nerve regeneration. In this work, chemical sympathectomy by 6-hydroxydopamine provided the model for assessing heart GDNF expression during denervation and axonal regrowth. A glyoxylic acid-based histochemical technique evaluated the noradrenergic innervation. ELISA determined GDNF levels after concentrating heart homogenates. Light and ultrastructural in situ hybridization and immunocytochemistry were used for identifying cells expressing GDNF mRNA and protein. In control rats, the GDNF cardiac levels were significantly higher in 37-day-old animals in comparison with those aging 60 days. In sympathectomized rats, GDNF cardiac levels were significantly higher 7 days after sympathectomy and dropped to control levels at day 30. GDNF mRNA was expressed in atrial and ventricular myocytes from normal and sympathectomized rats. GDNF immunoreactivity occurred on atrial granules and quantitative analysis in electron micrographs confirmed ELISA-obtained data. In ventricular myocytes gold particles occurred sparsely. These findings constitute the first evidence for GDNF synthesis by cardiomyocytes and postulate a role for this factor soon after cardiac sympathetic denervation, probably in nerve regeneration. In atrial myocytes, GDNF is probably secreted by regulated pathway.