M3 subtype of muscarinic acetylcholine receptor promotes cardioprotection via the suppression of miR-376b-5p

The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.     

Choline produces cytoprotective effects against ischemic myocardial injuries: evidence for the role of cardiac m3 subtype muscarinic acetylcholine receptors.

BACKGROUND/AIMS: Accumulating evidence indicates the presence of functional M3 subtype of acetylcholine muscarinic receptors (M(3)-mAChR), in addition to the well-recognized M(2)-mAChR, in the heart of various species including man. However, the pathophysiological role of the cardiac M(3)-mAChR remain undefined. This study was designed to explore the possible role of M(3)-mAChR in cytoprotection of myocardial infarction and several related signaling pathways as potential mechanisms. METHODS: Studies were performed in a rat model of myocardial infarction and in isolated myocytes. RESULTS: We found that choline relieved myocardial injuries during ischemia or under oxidative stress, which was achieved by correcting hemodynamic impairment, diminishing ventricular arrhythmias and protecting cardiomyocytes from apoptotic death. The beneficial effects of choline were reversed by the M(3)-selective antagonists but not by the M(2)-selective antagonist. Choline/M(3)-mAChR activated several survival signaling molecules (antiapoptotic proteins Bcl-2 and ERKs), increased endogenous antioxidant reserve (SOD), and reduced apoptotic mediators (proapoptotic proteins Fas and p38 MAPK) and intracellular Ca2+ overload. CONCLUSION: Choline improves cardiac function and reduces ischemic myocardial injuries via stimulating the cardiac M(3)-mAChRs which in turn result in alterations of multiple signaling pathways leading to cytoprotection. The findings suggest M(3)-mAChR as a new target for drug development for improving cardiac function and preventing cardiac injuries during ischemia/reperfusion.     

G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes

This study tests the hypothesis that G-protein-coupled receptor (GPCR) signaling components involved in the regulation of adenylyl cyclase (AC) localize with caveolin (Cav), a protein marker for caveolae, in both cell-surface and intracellular membrane regions. Using sucrose density fractionation of adult cardiac myocytes, we detected Cav-3 in both buoyant membrane fractions (BF) and heavy/non-buoyant fractions (HF); beta2-adrenergic receptors (AR) in BF; and AC5/6, beta1-AR, M4-muscarinic acetylcholine receptors (mAChR), mu-opioid receptors, and Galpha(s) in both BF and HF. In contrast, M2-mAChR, Galpha(i3), and Galpha(i2) were found only in HF. Immunofluorescence microscopy showed co-localization of Cav-3 with AC5/6, Galpha(s), beta2-AR, and mu-opioid receptors in both sarcolemmal and intracellular membranes, whereas M2-mAChR were detected only intracellularly. Immunofluorescence of adult heart revealed a distribution of Cav-3 identical to that in isolated adult cardiac myocytes. Upon immunoelectron microscopy, Cav-3 co-localized with AC5/6 and Galpha(s) in sarcolemmal and intracellular vesicles, the latter closely allied with T-tubules. Cav-3 immunoprecipitates possessed components that were necessary and sufficient for GPCR agonist-promoted stimulation and inhibition of cAMP formation. The distribution of GPCR, G-proteins, and AC with Cav-3 in both sarcolemmal and intracellular T-tubule-associated regions indicates the existence of multiple Cav-3-localized cellular microdomains for signaling by hormones and drugs in the heart.     

Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia

Connexin-protein interactions are believed to be critical for the regulation of gap junctional intercellular communication and for the function of gap junctions formed by these complexes. We have primarily used immunoprecipitation strategies to investigate whether connexin43 binds to selected signaling and cytoskeletal proteins and whether connexin43-protein binding is altered in cultured astrocytes exposed to chemical ischemia/hypoxia, a treatment that resembles ischemia in vivo. Chemical ischemia/hypoxia induced marked dephosphorylation of connexin43, which was accompanied by increased association of connexin43 with c-Src, ERK1/2, and mitogen-activated protein kinase phosphatase-1 and by decreased association between connexin43 and beta-actin. Moreover, we found that endogenous c-Src in normal astrocytes exists primarily in the Triton X-100-soluble membrane fraction, distinct from the Triton-insoluble fraction, which contains gap junctions. After chemical ischemia/hypoxia, c-Src appeared in the Triton-insoluble fraction and was co-immunoprecipitated with connexin43, suggesting that chemical ischemia/hypoxia induced translocation of c-Src to the Triton-insoluble fraction and association with connexin43. Furthermore, the "dephosphorylated" form of connexin43 was immunoprecipitated by a phosphotyrosine antibody, suggesting tyrosine phosphorylation of connexin43 by c-Src. In addition, the association between connexin43 and c-Src was blocked by inhibition of connexin43 dephosphorylation, suggesting that the interaction between connexin43 and c-Src can be regulated by alterations in the phosphorylation state of connexin43. These results identify new binding partners for connexin43 and demonstrate that interactions between connexin43 and protein kinases and phosphatases are dynamically altered as a consequence of connexin43 phosphorylation.     

Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.     

Expression and Agonist-Induced Down-Regulation of mRNAs of m2- and m3-Muscarinic Acetylcholine Receptors in Cultured Cerebellar Granule Cells

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.     

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).     

Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium

The activity of the Na(+)/H(+) exchanger has been implicated as an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the protein in ischemic and reperfused isolated hearts and isolated ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the exchanger after recovery from ischemia. Ischemia followed by reperfusion caused a strong inhibitory effect on NHE1 activity that abated with continued reperfusion. Four major protein kinases of size 90, 55, 44, and 40 kDa phosphorylated the Na(+)/H(+) exchanger. The Na(+)/H(+) exchanger-directed kinases demonstrated dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and isolated myocytes. p90(rsk) was identified as the 90-kDa protein kinase activated by ischemia and reperfusion while ERK1/2 was identified as accounting for some of the 44-kDa protein kinase phosphorylating the Na(+)/H(+) exchanger. The results demonstrate that MAPK-dependent pathways including p90(rsk) and ERK1/2 and are important in regulating the Na(+)/H(+) exchanger and show their dramatic increase in activity toward the Na(+)/H(+) exchanger during ischemia and reperfusion of the myocardium. They also show that ischemia followed by reperfusion have important inhibitory effects on Na(+)/H(+) exchanger activity.     

Studies on the Characterization of the Subtype(S) of Muscarinic Receptor Involved in Prostacyclin Synthesis in Rabbit Cardiomyocytes

Abstract

The present study was conducted to localize and characterize the subtype(s) of muscarinic receptor involved in prostacyclin (PGI₂) production elicited by the cholinergic transmitter acetylcholine (ACh) in various cell types in the rabbit heart. ACh increased PGI₂ synthesis measured as 6-keto-PGF1α, in cultured coronary endothelial cells and freshly dissociated ventricular myocytes in a dose dependent manner but not in cultured coronary smooth muscle cells of rabbit heart. McN-A-343, a partially selective M₁ muscarinic ACh receptor (mAChR) agonist, did not alter 6-keto-PGF1α synthesis in these cell types. ACh induced 6-keto-PGF1α synthesis in coronary endothelial cells and ventricular myocytes was not altered by a low concentration (10^?8 M) of pirenzipine, an M₁ mAChR antagonist but was reduced by a higher concentration (10^?6 M). In coronary endothelial cells ACh induced 6-keto-PGF1α production was reduced by hexahydro-sila-difendial (HHSiD), an M₃ mAChR antagonist, and in ventricular myocytes by both 11-(2-[(di-ethylamino) methyl]-1-piperidinyl]acetyl-5,11-dihydro-6-H-pyrido-[2,3-b]-benzodiazepine-6 one] (AF-DX 116), an M₂ receptor antagonist, and HHSiD. The decrease by ACh of isoporterenol stimulated cAMP accumulation was minimized by AF-DX 116 but not by HHSiD or pirenzipine. Pertussis toxin treatment minimized ACh induced decrease in isoproterenol stimulated rise in cAMP and ATP release, but not ACh induced 6-keto-PGF1α synthesis. These data suggest that ACh stimulates prostacyclin production in coronary endothelial cells via M₃ mAChR and in ventricular myocytes M₂ and M₃ mAChR. Moreover, ACh induced decrease in cAMP, but not the increase in 6-keto-PGF1α production, is mediated by pertussis toxin sensitive Gαi proteins in these cells.     

M3 muscarinic receptor activation of a delayed rectifier potassium current in canine atrial myocytes.

Growing body of evidence indicates that the functional responses of cells to muscarinic acetylcholine receptors (mAChRs) are mediated by multiple receptor subtypes. It is commonly thought that the M2 receptor is the only functional mAChR subtype in the heart and little data regarding the potential roles of other subtypes in cardiac tissues has been reported. In the present study, we provide functional evidence for the presence and physiological function of an M3 receptor in canine atrial myocytes. Using whole-cell patch-clamp techniques, we consistently found that pilocarpine, an mAChR agonist, induced a K+ current similar to but distinct from the classical delayed rectifier K+ current. Same observations were obtained when choline or tetramethylammonium (TMA) was applied to the bath. The currents were abolished by 1 microM atropine. Antagonists selective to M1 (pirenzepine, 100 nM), M2 (methoctramine 100 nM), or M4 (tropicamide 200 nM) receptors failed to alter the currents. Conversely, three different M3-selective inhibitors, p-F-HHSiD (20-200 nM), 4-DAMP methiodide (2-10 nM) and 4-DAMP mustard (4-20 nM), all produced concentration-dependent suppression of the currents. A cDNA fragment representing the M3 receptor was isolated from dog atrial RNA and the mRNA level of this construct was 0.7 +/- 0.1 pg/microg total RNA, as quantified by the competitive RT-PCR methods. Our data strongly suggested that an M3 receptor exists and is coupled to a K+ channel in the heart.     

The reliability of the ankle-brachial index in the Atherosclerosis Risk in Communities (ARIC) study and the NHLBI Family Heart Study (FHS)

Background

Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro.

Methods

To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin^® and the polycation transduction enhancer Transfectam^®. The EGFP-positive transduced cells were then enriched by flow cytometry.

Results

More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes.

Conclusion

The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.     

Maintenance of intercellular coupling by the antiarrhythmic peptide rotigaptide suppresses arrhythmogenic discordant alternans

Discordant action potential alternans creates large gradients of refractoriness, which are thought to be the mechanisms linking T-wave alternans to cardiac arrhythmogenesis. Since intercellular coupling acts to maintain synchronization of repolarization between cells, we hypothesized that intercellular uncoupling, such as during ischemia, would initiate discordant alternans and that restoration of intercellular coupling by the gap junction opener rotigaptide may provide a novel approach for suppressing arrhythmogenic discordant alternans. Optical mapping was used to record action potentials from ventricular epicardium of Langendorff-perfused guinea pig hearts. Threshold for spatially synchronized (i.e., concordant) alternans and discordant alternans was determined by increasing heart rate step-wise during 1) baseline, 2) treatment with rotigaptide or vehicle, and 3) global low-flow ischemia + rotigaptide or vehicle. Ischemia reduced the threshold for concordant alternans in both groups from 362 +/- 8 to 305 +/- 9 beats/min (P < 0.01) and for discordant alternans from 423 +/- 6 to 381 +/- 7 beats/min (P < 0.01). Interestingly, rotigaptide also increased the threshold for discordant alternans relative to vehicle both before (438 +/- 7 vs. 407 +/- 8 beats/min, P < 0.05) and during (394 +/- 7 vs. 364 +/- 9 beats/min, P < 0.05) ischemia. Rotigaptide increased conduction velocity and prevented conduction slowing and dispersion of repolarization during ischemia. Confocal immunofluorescence revealed that total connexin43 quantity and cellular distribution were unchanged before or after low-flow ischemia, with and without rotigaptide. However, connexin43 dephosphorylation in response to low-flow ischemia was significantly prevented by rotigaptide (15.9 +/- 7.0 vs. 0.3 +/- 6.4%, P < 0.001). These data suggest that intercellular uncoupling plays an important role in the transition from concordant to discordant alternans. By suppressing discordant alternans, repolarization gradients, and connexinx43 dephosphorylation, rotigaptide may protect against ischemia-induced arrhythmias. Drugs that selectively open gap junctions offer a novel strategy for antiarrhythmic therapy.     

Coexpression of connexin 45 with connexin 43 decreases gap junction size

In the human heart, ventricular myocytes express connexin 43 (Cx43) and traces of Cx45. In congestive heart failure, Cx43 levels decrease, Cx45 levels increase and gap junction size decreases. To determine whether alterations of connexin coexpression ratio influence gap junction size, we engineered a rat liver epithelial cell line that endogenously expresses Cx43 to coexpress inducible levels of Cx45 under stimulation of the insect hormone, ponasterone A. In cells induced to express Cx45, gap junction sizes are significantly reduced (by 15% to 20%; p < 0.001), an effect that occurs despite increased levels of junctional connexons made from both connexins. In contrast, coexpression of Cx40 with Cx43 does not lead to any change in gap junction size. These results are consistent with the idea that increased Cx45 expression in the failing ventricle contributes to decreased gap junction size.     

Acetylcholine stimulates cyclic ADP-ribose formation via M1 muscarinic receptors in rat superior cervical ganglion

The role of cyclic ADP-ribose (cADPR) as the downstream signal of neuronal muscarinic acetylcholine receptors (mAChRs) and the enzyme responsible for its synthesis, ADP-ribosyl cyclase, were examined in the rat superior cervical ganglion (SCG). Application of acetylcholine or other mAChR agonists increased the ADP-ribosyl cyclase activity by about 250-300% in crude membrane fractions from the SCG of 14-day-old rats. This effect was inhibited by atropine or by the M1-mAChR antagonist, pirenzepine, and was mimicked by GTP. These results indicate that the M1 mAChRs couple to the membrane-bound form of ADP-ribosyl cyclase and suggest that cADPR is a second messenger of M1 mAChR signaling in nervous tissue.     

Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border.

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.     

Activation of calcineurin expression in ischemia-reperfused rat heart and in human ischemic myocardium

Calcineurin (CaN) has been reported as a critical mediator for cardiac hypertrophy and cardiac myocyte apoptosis. In the present study, we investigated the activity and expression of CaN and the effect of calpain in rat heart after ischemia and reperfusion. Rat ischemic heart showed significant increase in CaN activity. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine CaN indicated a prominent immunoreactive band of 60 kDa (CaN A). In ischemic-reperfused hearts, the expression of CaN A was significantly low and immunoreactivity was observed in proteolytic bands of 46 kDa. This may be due to the proteolytic degradation of CaN A in ischemic tissues by m-calpain. We also noticed in vitro proteolysis of bovine cardiac CaN A by m-calpain. Immunohistochemical studies showed strong staining of immunoreactivity in rat hearts that had gone under 30 min ischemia followed by 30 min reperfusion similar to that found in human ischemic heart. Ischemia is associated with multiple alterations in the extracellular and intracellular signaling of cardiomyocytes and may act as an inducer of apoptosis. The increase in CaN activity and strong immunostaining observed in ischemic/perfused rat heart may be due to the calpain-mediated proteolysis of this phosphatase.     

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/-; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/- myocytes had significantly shorter action potential durations (APD) and increased steady-state (Iss) and inward rectifier (I(K1)) potassium currents compared with those of wild-type littermate cells. In Cx43+/- hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/- hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.