Kuan-Cheng Chang*†, Andreas S. Barth*, Tetsuo Sasano*, Eddy Kizana*, Yuji Kashiwakura*, Yiqiang Zhang*, D. Brian Foster*, and Eduardo Marba´n*‡§
*Institute of Molecular Cardiobiology, The Johns Hopkins University, Baltimore, MD 21205;†Graduate Institute of Clinical Medical Science, China Medical University and Hospital, Taichung 40447, Taiwan; and‡Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 10, 2008 (received for review September 26, 2007)
Congenital long- or short-QT syndrome may lead to life-threatening ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in CAPON, a neuronal nitric oxide synthase (NOS1) regulator, have recently been associated with QT interval variations in a human whole-genome association study. CAPON had been unsuspected of playing a role in cardiac repolarization; indeed, its physiological role in the heart (if any) is unknown. To define the biological effects of CAPON in the heart, we investigated endogenous CAPON protein expression and protein–protein interactions in the heart and performed electrophysiological studies in isolated ventricular myocytes with and without CAPON overexpression. We find that CAPON protein is expressed in the heart and interacts with NOS1 to accelerate cardiac repolarization by inhibition of L-type calcium channel. Our findings provide a rationale for the association of CAPON gene variants with extremes of the QT interval in human populations.
NOS1兩 QT interval 兩 cardiac electrophysiology
R
are disease-causing mutations leading to congenital long- or short-QT syndrome are well recognized, but there is little insight into genetic sources of QT interval variation in normal populations. A whole-genome association approach has recently implicated common genetic variants in CAPON as contributing to QT interval differences in a community-based German population (1). This association has since been confirmed in other populations (2, 3). The genetic findings challenge our current understanding of QT physiology. CAPON, first identi-fied in rat brain neurons (4), is a highly conserved protein (⬇92% conceptual amino acid sequence identity between rat and human) with an N-terminal phosphotyrosine-binding (PTB) domain and a C-terminal PDZ-binding [postsynaptic density-95 (PSD95)/drosophila discs large/zona occludens-1] domain (4–6).In brain, CAPON competes with PSD95 for the binding of neuronal nitric oxide synthase (NOS1) through the interaction of its C terminus with the PDZ domain of NOS1 (4), thus uncou-pling the NMDA–NOS1–NO-mediated signaling pathways.
CAPON is also an adaptor protein of NOS1, capable of directing NOS1 to specific target proteins (5, 6). Nowhere, however, has CAPON been suspected of playing a role in cardiac physiology.
Both NOS1 and endothelial NOS (NOS3) are constitutively expressed in cardiomyocytes (7). NOS1 in the sarcolemma has been proposed to interact with Na⫹-K⫹ATPase (8) and with the plasma membrane Ca2⫹/calmodulin-dependent Ca2⫹ ATPase (PMCA) through the interaction of PDZ domain of NOS1 and the C terminus of PMCA4b isoform (9). In the sarcoplasmic reticulum (SR), NOS1 is structurally associated with ryanodine receptor 2 (RyR2) (10) and cardiac SR Ca2⫹ ATPase (SERCA2) (11) to regulate intracellular calcium cycling and excitation–contraction coupling. Conditional transgenic overexpression of NOS1 in heart leads to additional association of NOS1 and the sarcolemmal L-type calcium channel and thus suppresses the L-type calcium currents
(ICa,L) (12). All of these lines of evidence highlight the notion that NOS1 plays a key role in regulating cardiac physiology.
Here, we hypothesized that CAPON is expressed in the heart and interacts with NOS1 to exert its biological effects. Therefore, we first sought to identify endogenous CAPON protein in ventricular myocytes and to characterize its physiological relevance by over-expression of CAPON usingin vivo somatic gene transfer. We then probed the interaction between CAPON and the NOS–NO signal-ing pathways to dissect the potential mechanisms underlysignal-ing the biological effects of CAPON in the heart.
Results
Identification of Endogenous CAPON Protein in the Heart.Because CAPON expression has not been documented in the heart, we first sought to probe the endogenous CAPON protein in guinea pig ventricular myocardium by Western blotting. The protein bands from guinea pig heart were compared with those from lung and brain tissues and from HEK293 cells with heterologous overex-pression of CAPON by in vitro gene transfer with a bicistronic adenoviral vector (AdCAPON-GFP). The ventricular myocardium exhibited a band migrating near the 60-kDa marker, as expected for endogenous full-length CAPON, comparable with the comigration bands from HEK293 cells with heterologously overexpressed CAPON and from brain, which has abundant endogenous CAPON (Fig. 1A). The higher bands (⬇70 kDa) could reflect either posttranslational modification of CAPON or nonspecific cross-reactivity. Notably, the ventricular myocardium also displayed a band⬇30 kDa in size detected by antibody against the C terminus of CAPON, but not by antibody against an N-terminal epitope, which is consistent with the short-form of CAPON (13). Immuno-fluorescent staining of freshly isolated ventricular myocytes re-vealed a cytoplasmic distribution pattern of the soluble CAPON protein with focal accentuation over the perinuclear region and the sarcolemmal membrane (Fig. 1B). Additionally, enhancement of CAPON immunofluorescence can be appreciated in CAPON-overexpressing myocytes. Therefore, expression of the endogenous CAPON protein in ventricular myocytes and overexpression of CAPON by AdCAPON-GFP-mediated gene transfer were both confirmed biochemically.
Adenovirus Alone Does Not Affect Electrophysiology.Both action potential duration (APD) and peak ICa,Ldensity were equivalent between AdGFP-transduced and nontransduced myocytes (Fig. 2
Author contributions: E.M. designed research; K.-C.C., A.S.B., T.S., E.K., Y.K., and Y.Z.
performed research; T.S., E.K., Y.K., and D.B.F. contributed new reagents/analytic tools;
K.-C.C., A.S.B., T.S., E.K., Y.K., Y.Z., D.B.F., and E.M. analyzed data; and K.-C.C., A.S.B., and E.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
§To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online atwww.pnas.org/cgi/content/full/
0709118105/DC1.
© 2008 by The National Academy of Sciences of the USA
PHYSIOLOGY
A and B). Seesupporting information (SI)Resultsfor details. After ascertaining that adenoviral transduction alone does not alter cellular electrophysiology, we examined the effects of CAPON overexpression by comparing the electrophysiological parame-ters between AdCAPON-GFP-transduced and nontransduced myocytes.
CAPON Overexpression Accelerates Action Potential.To determine whether CAPON modulates cardiac repolarization, we compared the APD in freshly isolated control and CAPON-overexpressing ventricular myocytes at 1, 2, and 0.05 Hz. At 1-Hz stimulation, the mean APD10, APD50, APD75, and APD90were significantly short-ened to 54.0⫾ 7.2, 198.8 ⫾ 8.1, 221.8 ⫾ 7.9, and 233.9 ⫾ 7.7 ms in CAPON-overexpressing cardiomyocytes (n ⫽ 9) compared with 96.6⫾ 11.5, 291.0 ⫾ 16.4, 316.1 ⫾ 17.2, and 327.5 ⫾ 17.9 ms in control myocytes (n ⫽ 13, P ⬍ 0.05, respectively) (Fig. 2 C–E). The abbreviation of APD with CAPON overexpression was maintained at 2 Hz and 0.05 Hz (data not shown), and the magnitude of APD90
reduction by CAPON overexpression was 28.6% at 1-Hz stimula-tion. We next explored the ionic basis for the abbreviation of APD.
L-Type Calcium Current.In CAPON-overexpressing myocytes, the peak ICa,Ldensity was significantly reduced (⫺7.2 ⫾ 0.5 pA/pF, at
⫹20 mV, n ⫽ 8) compared with that of control myocytes (⫺11.0 ⫾ 1.0 pA/pF, at⫹10 mV, n ⫽ 12; P ⬍ 0.05) (Fig. 2 F and G). The reduction of the peak ICa,Ldensity was 35% (Fig. 2G). Averaged peak current density–voltage relationships revealed significant sup-pression of peak ICa,Ldensity from⫺30 mV to ⫹40 mV (P ⬍ 0.05, respectively) by CAPON overexpression (Fig. 2H). By a decrease in net inward current, the CAPON-induced inhibition of peak ICa,L
density would contribute to the abbreviation of APD.
Sodium Current.Sodium current (INa) was not affected by CAPON overexpression (Fig. 3A and B). For details seeSI Results.
Outward Rectifier Potassium Current. In guinea pig ventricular myocytes, there are two types of delayed rectifier potassium cur-rents, the rapidly activating component (IKr) and the slowly acti-vating component (IKs) (14), responsible for terminating the action potential plateau. To probe the potential contribution of these currents to CAPON overexpression-mediated APD changes, we first recorded the total IKtail currents and then used a specific IKs
blocker, chromanol 293B (15), to separate IKrand IKsin CAPON-overexpressing and control myocytes. Although neither the total IK
nor the IKstail current density was changed between nontransduced control and CAPON-overexpressing myocytes, we found the IKr
peak tail current density to be modestly enhanced in CAPON-overexpressing myocytes (0.92⫾ 0.07 pA/pF, n ⫽ 10) compared with control myocytes (0.61⫾ 0.08 pA/pF, n ⫽ 7; P ⬍ 0.05) (Fig.
3C and D). These results indicate that in addition to the reduction of ICa,L, enhancement of IKralso contributes to the abbreviation of APD in CAPON-overexpressing myocytes.
Inward Rectifier Potassium Current.Inward rectifier potassium cur-rent (IK1) was not changed by CAPON overexpression (Fig. 3E).
SeeSI Resultsfor details.
Protein–Protein Interaction of CAPON and NOS1 in the Heart.After identifying that both full-length and short-form CAPON proteins are expressed in the heart and overexpression of CAPON accel-erates APD by suppression of ICa,Land augmentation of IKr, the important next step is to answer whether the electrophysiological changes induced by CAPON overexpression were caused by mod-ifications of the NOS–NO pathways. First, we determined whether
80
Fig. 1. Identification of endogenous CAPON protein in the heart. (A) Tissue homogenates from normal guinea pig ventricular myocardium (VM), brain, and lung, and HEK293 cells with (T) and without (NT) in vitro transduction with AdCAPON-GFP were subjected to SDS/PAGE Western blotting. Both full-length (CAPON-L) and short-form of CAPON (CAPON-S) are expressed in VM.
The higher bands (arrowhead) could be caused by either posttranslational modification of CAPON or nonspecific cross-reactivity. (B) Immunostaining of CAPON in AdCAPON-GFP-transduced (3) and nontransduced (2) ventricular myocytes. Negative controls (secondary antibody only) are depicted in 1.
1 Hz Peak ICa,L (pA/pF)
CAPON-1 Hz
Fig. 2. Electrophysiological effects of CAPON overexpression in guinea pig ventricular myocytes. (A and B) Adenovirus alone does not affect electrophysiology. For details, seeSI Results.
(C–H) CAPON overexpression abbreviates the action potential duration and reduces ICa,L. (C) APD was markedly shortened in a representative AdCAPON-GFP-transduced myocyte com-pared with a representative control myocyte. (D) Significant abbreviation of APD can be seen spanning from APD10to APD90at 1-Hz stimulation in CAPON-overexpressing myocytes (CAPON, n⫽ 9; control, n ⫽ 13). (E) The actual APD50and APD90
measured from individual myocyte comprising each group reveal a consistent reduction of APD in CAPON-overexpressing myocytes. (F) Representative ICa,Lrecordings elicited by depo-larizing voltage steps (500 ms) from⫺40 to ⫹60 mV in 10-mV increments after a prepulse from⫺80 mV to ⫺40 mV show reduction of ICa,Lin a AdCAPON-GFP-transduced myocyte com-pared with a control myocyte. (G) The peak ICa,Ldensity in CAPON-overexpressing myocytes was smaller than in control myocytes. (H) Averaged peak current–voltage relationships demonstrate attenuation of ICa,Lat multiple depolarizing pulses in CAPON-overexpressing myocytes (CAPON, n⫽ 8;
control, n⫽ 12). The number inside each bar graph indicates the number of cells studied.
CAPON interacts with NOS in the heart. To assess protein–protein interactions of CAPON and NOS, normal guinea pig ventricular tissue homogenates were immunoprecipitated with anti-CAPON-or anti-NOS1-bound protein G complex, separated by SDS/PAGE, and probed by CAPON, NOS1, and NOS3 antibodies, respectively.
The crude tissue homogenates served as the positive controls while probing NOS1, NOS3, and CAPON (Fig. 4). We found that NOS1 specifically coimmunoprecipitated with the bound CAPON anti-bodies (Fig. 4A and B), whereas NOS3 was not detected in the
anti-CAPON immunoprecipitates (Fig. 4C and D). To include a negative control, the ventricular myocardial homogenates were also incubated overnight with the protein G complex without prebound CAPON antibody; as a result, neither NOS1 nor CAPON could be detected in the eluted precipitates (Fig. 4A and B). These findings indicate that CAPON–NOS1, but not CAPON–NOS3, exists as a physiological complex in ventricular myocytes.
CAPON Overexpression Stabilizes NOS1.Because CAPON interacts with NOS1 in the heart, we next wanted to know how CAPON overexpression affects NOS1 protein level and activity. Freshly isolated ventricular myocytes were transduced in vitro with AdCAPON-GFP, or AdGFP, or not transduced with either virus.
The gene transfer efficiency was confirmed by ⬇100% GFP-positive cells in AdGFP-transduced myocytes and ⬇50% GFP positive cells in AdCAPON-GFP myocytes, compared with only background autofluorescence (i.e., 0% GFP positivity) in nontrans-duced myocytes (Fig. 5A). The in vitro AdCAPON-GFP transduc-tion caused a 1.9-fold increase of the CAPON protein level (Fig.
5B). At 0 h of culture, the NOS1 level was not different between AdCAPON-GFP-transduced and nontransduced myocytes (5.47⫾ 2.51% vs. 6.08⫾ 2.27%, n ⫽ 3 animals; p ⫽ NS) (Fig. 5C). After 40.3⫾ 2.3 h of cell culture, NOS1 was down-regulated in nontrans-duced and AdGFP-transnontrans-duced myocytes but was up-regulated in AdCAPON-GFP-transduced myocytes. Therefore, the mean NOS1 level from nontransduced and AdGFP-transduced myocytes was lower than that of AdCAPON-GFP-transduced myocytes (2.78⫾ 1.75% vs. 18.54 ⫾ 5.99%; P ⬍ 0.05) (Fig. 5D). Of note, NOS3 protein levels were not changed in AdCAPON-GFP-transduced myocytes (data not shown). Noncultured ventricular tissue homogenates served as positive control for the NOS1/NOS3
-80-70-60-50-40-30-20-10 10 20
Peak Tail Current (pA/pF)
A B
C D
Fig. 3. Electrophysiological effects of CAPON overexpression in guinea pig ventricular myocytes. (A and B) CAPON overexpression does not affect sodium current. For more details, seeSI Results. (C–E) CAPON overexpression enhances IKr. (C) Delayed rectifier K⫹tail current was measured in response to a depo-larizing pulse to⫹40 mV for 5 s followed by repolarization to ⫺40 mV. IKrwas recorded after steady-state suppression of IKsby chromanol 293B. Represen-tative recordings show a larger IKrtail current in a CAPON-overexpressing myocyte compared with a control myocyte. (D) Neither peak IKnor IKstail current densities were significantly different between CAPON-overexpressing and control myocytes. However, peak IKrtail current density was modestly enhanced in CAPON-overexpressing myocytes. (E) Instantaneous IK1current density elicited by ramp protocol from⫺100 to ⫹ 70 mV (5 s) was not different between CAPON-overexpressing (n⫽ 10) and control myocytes (n ⫽ 9).
IP-CAPON
Fig. 4. CAPON interacts with NOS1, but not NOS3, in ventricular myocytes.
Normal guinea pig ventricular tissue homogenates were immunoprecipitated overnight with anti-CAPON-bound protein G complex, separated by SDS/
PAGE, and probed by anti-NOS1 (A), anti-CAPON (B and D), and anti-NOS3 (C), respectively. To include a negative control, the ventricular tissue homoge-nates were also incubated overnight with anti-CAPON-free protein G complex (beads). As a result, neither NOS1 nor CAPON could be detected in the eluted precipitates. WB, Western blotting.
BF GFP
No virus AdCAPON-GFP VM
D
Culture (Hrs) 40.3± 2.3
*
Fig. 5. CAPON overexpression stabilizes NOS1 in ventricular myocytes. (A) Freshly isolated ventricular myocytes were transduced in vitro with AdCAPON-GFP, or AdAdCAPON-GFP, or not transduced with either virus. BF, bright-field microscopic images. (B) The in vitro AdCAPON-GFP transduction caused a 1.9-fold increase of the CAPON level. (C) At 0 h of culture, the NOS1 level was not different between AdCAPON-GFP-transduced and nontransduced myocytes (n⫽ 3 animals). (D) After 40.3⫾ 2.3 h of cell culture, NOS1 became down-regulated in both ventricular myocytes transduced with AdGFP and in nontransduced myocytes, whereas NOS1 was well preserved in the AdCAPON-GFP-transduced ventricular myocytes. The mean NOS1 level from nontransduced and AdGFP-transduced myocytes was lower than that of AdCAPON-GFP-AdGFP-transduced myo-cytes (*, P⬍ 0.05 vs. control, n ⫽ 3 animals). Noncultured ventricular tissue homogenates (VM) served as positive control for NOS1 detection. The signal intensities of CAPON and NOS1 are normalized against the GAPDH signals.
PHYSIOLOGY
expression assays. These findings suggest that CAPON overexpres-sion may stabilize NOS1 in ventricular myocytes.
Enhanced Intracellular NO Production in CAPON-Overexpressing Myo-cytes.Because NO generation reflects NOS activity, we further imaged intracellular NO production by using a rhodamine-based chromophore, DAR-4M AM, in living ventricular myocytes iso-lated 3–5 days afterin vivo gene transfer of CAPON. To test the specificity of DAR-4M AM for NO imaging, we measured
fluo-rescence intensity in ventricular myocytes loaded with DAR-4M AM only or DAR-4M AM⫹ sodium nitroprusside (SNP) or DAR-4M AM ⫹ NG-nitro-L-arginine methyl ester (L-NAME).
Fluorescence intensified with the addition of SNP and waned with
L-NAME compared with that in myocytes incubated with DAR-4M AM only (Fig. 6A). Next, we compared NO production between CAPON-overexpressing myocytes and control myocytes incubated with DAR-4M AM only or DAR-4M AM⫹ 2 mM
L-arginine (Fig. 6 B–D). The baseline fluorescent intensity was equivalent in CAPON-overexpressing and control myocytes (921.6⫾ 32.5 a.u., n ⫽ 30 vs. 888.6 ⫾ 17.3 a.u., n ⫽ 90; P ⫽ NS), whereas with the addition ofL-arginine, the fluorescence intensity increased disproportionately in CAPON-overexpressing myocytes (1,420.9⫾ 28.1 a.u., n ⫽ 43) compared with controls (1,329.0 ⫾ 19.1 a.u.,n ⫽ 166; P ⬍ 0.05) (Fig. 6D). These results indicate that CAPON overexpression may enhance intracellular NO production, particularly in the presence of additional NOS substrates.
L-NAME Reverses CAPON Overexpression-Mediated APD Abbreviation and ICa,LReduction.After ascertaining that CAPON overexpression resulted in up-regulation of NOS1–NO activity, we examined the effects ofL-NAME on ICa,L and APD in ventricular myocytes isolated 3–5 days afterin vivo gene transfer of CAPON. Pretreat-ment with 1 mML-NAME reversed the APD90abbreviation (1 Hz, 348.3⫾ 47.8 ms, n ⫽ 7 vs. 378.5 ⫾ 50.1 ms, n ⫽ 5; P ⫽ NS) (Fig.
7A and B) and the ICa,Ldensity reduction (⫺6.2 ⫾ 0.6 pA/pF, n ⫽ 14 vs. ⫺6.6 ⫾ 0.4 pA/pF, n ⫽ 14; P ⫽ NS) (Fig. 7 C–E) in CAPON-overexpressing ventricular myocytes. Interestingly, APD90tends to become longer (378.5⫾ 50.1 ms, n ⫽ 5 vs. 333.7 ⫾ 20.2 ms,n ⫽ 7; P ⫽ NS) (Fig. 7 A and B), and the peak ICa,Ldensity tends to increase, too (⫺6.6 ⫾ 0.4 pA/pF, n ⫽ 14 vs. ⫺6.3 ⫾ 0.3 pA/pF,n ⫽ 14; P ⫽ NS) (Fig. 7 C–E) in control myocytes after pretreatment with L-NAME. However, these changes were not statistically significant. In contrast, significant lengthening of APD90(1 Hz, 348.3⫾ 47.8 ms, n ⫽ 7 vs. 221.6 ⫾ 19.9 ms, n ⫽ 6;
P ⬍ 0.05) (Fig. 7 A and B) and the increase of the peak ICa,Ldensity (⫺6.2 ⫾ 0.6 pA/pF, n ⫽ 14 vs. ⫺4.8 ⫾ 0.3 pA/pF, n ⫽ 14; P ⬍ 0.05) (Fig. 7C–E) were only seen in CAPON-overexpressing myocytes after pretreatment withL-NAME.
Discussion
We demonstrate that CAPON, the brain-enriched NOS1 adaptor/
regulator protein, is expressed in the heart and interacts with NOS1.
DAR-4M AM
Fig. 6. Enhanced intracellular NO production in CAPON-overexpressing myocytes. (A) The specificity of DAR-4M AM was verified by a differential fluorescent intensity with the addition of SNP orL-NAME. (B) The NO fluores-cence was enhanced after L-arginine stimulation. In GFP (⫹), CAPON-overexpressing myocytes, the NO fluorescence was stronger than in control myocytes. (C) Representative high-powered images illustrate a modest en-hancement of the NO fluorescent marker in a CAPON-overexpressing myo-cyte. (D) The baseline fluorescent intensity was equivalent in CAPON-overexpressing (n⫽ 30) and control myocytes (n ⫽ 90), whereas with the addition ofL-arginine, the fluorescence intensity increased disproportionately in CAPON-overexpressing myocytes (CAPON-L-arg) (n⫽ 43) compared with controls (Control-L-arg) (n⫽ 166).
1 Hz
Peak ICa,L (pA/pF)
A B
C D
Fig. 7. L-NAME reverses CAPON overexpression-mediated APD abbreviation and ICa,Lreduction. (A) Representative action potential recordings show re-versal of APD abbreviation withL-NAME in a CAPON-overexpressing myocyte. (B) Pretreatment with
L-NAME significantly increased APD90 and reversed APD abbreviation in CAPON-overexpressing ventricu-lar myocytes, whereas the APD90 was not significantly changed withL-NAME in control myocytes. (C) Repre-sentative ICa,Lrecordings show reversal of ICa,L suppres-sion withL-NAME in a CAPON-overexpressing myo-cyte. (D) Averaged peak current–voltage relationships reveal significant increase of peak ICa,Ldensities with
L-NAME in CAPON-overexpressing myocytes but not in control myocytes. The number of cells studied in each group is indicated in bar graphs in E. (E) Pretreatment withL-NAME significantly increased peak ICa,Ldensity and rescued ICa,Lsuppression in CAPON-overexpress-ing myocytes, whereas the peak ICa,Ldensity was not significantly changed with L-NAME in control myocytes.
Overexpression of CAPON results in up-regulation of the NOS1–NO signaling pathways, which further leads to abbreviation of APD through the inhibition of ICa,Land enhancement of IKr. Expression of Endogenous CAPON Protein in the Heart.Since the first identification of CAPON protein in the rat brain (4), Xuet al. (13) further found that CAPON protein has two isoforms that are translated from a full-length and a C-terminal splicing variant (13).
The full-length transcript encompassing 10 exons contains both PTB- and PDZ-binding domains, whereas the C-terminal transcript containing the last 2 exons encodes only the PDZ-binding domain (13). We found that both full-length and short CAPON isoforms are expressed in guinea pig ventricular myocytes using the same CAPON antibody (13) predicted to react with the C terminus of CAPON. The full-length isoform comigrated at the same level as a brain-enriched protein band and a band from HEK293 cells expressing CAPON. Immunostaining reveals a cytoplasmic distri-bution of CAPON with focal accentuation around the perinuclear
The full-length transcript encompassing 10 exons contains both PTB- and PDZ-binding domains, whereas the C-terminal transcript containing the last 2 exons encodes only the PDZ-binding domain (13). We found that both full-length and short CAPON isoforms are expressed in guinea pig ventricular myocytes using the same CAPON antibody (13) predicted to react with the C terminus of CAPON. The full-length isoform comigrated at the same level as a brain-enriched protein band and a band from HEK293 cells expressing CAPON. Immunostaining reveals a cytoplasmic distri-bution of CAPON with focal accentuation around the perinuclear