Roles of PKC Isoforms in PMA-Induced Modulation of the hERG Channel (Kv11.1).

Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCε inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCε activator exhibited the opposite effect. Disruption of PKCß, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state (R(2)= 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.

Ramipril attenuates lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis.

INTRODUCTION: Recent studies revealed that co-morbidity and mortality due to cardiovascular disease are increased in patients with rheumatoid arthritis (RA) but little is known about factors involved in these manifestations. This study aimed at characterizing the impact of arthritis on oxidative stress status and tissue fibrosis in the heart of rats with adjuvant-induced arthritis (AIA). METHODS: AIA was induced with complete Freund's adjuvant in female Lewis rats. Animals were treated by oral administration of vehicle or angiotensin-converting enzyme inhibitor ramipril (10 mg/kg/day) for 28 days, beginning 1 day after arthritis induction. Isolated adult cardiomyocytes were exposed to 10 µM 4-hydroxynonenal (HNE) for 24 hours in the presence or absence of 10 µM ramipril. RESULTS: Compared to controls, AIA rats showed significant 55 and 30% increase of 4-HNE/protein adducts in serum and left ventricular (LV) tissues, respectively. Cardiac mitochondrial NADP+-isocitrate dehydrogenase (mNADP-ICDH) activity decreased by 25% in AIA rats without any changes in its protein and mRNA expression. The loss of mNADP-ICDH activity was correlated with enhanced accumulation of HNE/mNADP-ICDH adducts as well as with decrease of glutathione and NADPH. Angiotensin II type 1 receptor (AT1R) expression and tissue fibrosis were induced in LV tissues from AIA rats. In isolated cardiomyocytes, HNE significantly decreased mNADP-ICDH activity and enhanced type I collagen and connective tissue growth factor expression. The oral administration of ramipril significantly reduced HNE and AT1R levels and restored mNADP-ICDH activity and redox status in LV tissues of AIA rats. The protective effects of this drug were also evident from the decrease in arthritis scoring and inflammatory markers. CONCLUSION: Collectively, our findings disclosed that AIA induced oxidative stress and fibrosis in the heart. The fact that ramipril attenuates inflammation, oxidative stress and tissue fibrosis may provide a novel strategy to prevent heart diseases in RA.

Single intravenous low-dose injections of connexin 43 mimetic peptides protect ischemic heart in vivo against myocardial infarction.

The opening of unapposed connexin 43 hemichannels (Cx43Hc) under ischemic stress leads to cell death and irreversible tissue injury. Here, we investigate for the first time in vivo the cardioprotective potentials of two unique Cx43 structural-mimetic peptides (Cx43MPs) presumed specific blockers of Cx43Hc, Gap26 and Gap27, when injected intravenously using a rat model of myocardial infarction.Sprague Dawley rats were utilized. Myocardial infarction was induced by occluding the left anterior descending coronary for 40 min followed by 2 days of reperfusion. Interestingly, single bolus injections of Gap26 or Gap27 (1 µg/kg) into the jugular vein caused infarct size reductions by up to 61% with reference to control rats injected with saline at similar timings. Infarct reductions did not vary significantly whether peptides were administered before or after the onset of ischemia. Although the two peptides allegedly interact with distinct structures of Cx43, co-administration of Gap26/Gap27 in equal doses did not confer additive protection to hearts (maximum infarct reduction by 64%). Using patch clamp technique, we provide unique and direct evidence for the inhibitory effect of Cx43MPs on genuine human Cx43Hc transiently expressed in the ion channel-deficient tsA201 cells. In concordance with the cardioprotective effect observed in vivo, co-application of both peptides did not cause cumulative current inhibition. A safety profile of Cx43MPs was also addressed.Our results reveal great therapeutic potential of Cx43MPs in treatment of myocardial infarction. Their practical way and timing of administration and their apparent safe profile make them promising tools to fight ischemic heart disease.

Connexin 43 mimetic peptide Gap26 confers protection to intact heart against myocardial ischemia injury.

Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia-reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia-reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.

Phosphorylation of the consensus sites of protein kinase A on alpha1D L-type calcium channel.

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

Combined effects of reduced connexin 43, depressed active generator properties and energetic stress on conduction disturbances in canine failing myocardium.

To show that reductions in connexin43 (Cx43) can contribute, in association with electrophysiological alterations identified from unipolar recordings, to conduction disturbances in a realistic model of heart failure, canines were subjected to chronic rapid pacing (240/min for 4 weeks) and progressive occlusion of the left coronary circumflex artery (LCx) by an ameroid constrictor. Alterations identified from 191 epicardial recordings included abrupt activation delay, functional block, ST segment potential elevation, and reduced maximum negative slope (-dV/dt (max)). The LCx territory was divided into apical areas with depressed conduction velocity (LCx1: 0.06 +/- 0.04 m/s, mean +/- SD) and basal areas with relatively preserved conduction (LCx2: 0.28 +/- 0.01 m/s). Subepicardial Cx43 immunoblot measurements (percent of corresponding healthy heart measurements) were reduced in LCx1 ( approximately 40%) and LCx2 ( approximately 60%). In addition, -dV/dt (max) was significantly depressed (-3.8 +/- 3.3 mV/ms) and ST segment potential elevated (23.3 +/- 14.6 mV) in LCx1 compared to LCx2 (-9.5 +/- 3.4 mV/ms and 0.3 +/- 1.4 mV). Anisotropic conduction, Cx43 and ST segment potential measurements from the left anterior descending coronary artery territory, and interstitial collagen from all regions were similar to the healthy. Thus, moderate Cx43 reduction to "clinically relevant" levels can, in conjunction with regional energetic stress and depression of sarcolemmal active generator properties, provide a substrate for conduction disturbances.

Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site.

The Ca(v)1.3 (alpha(1D)) variant of L-type Ca(2+) channels plays a vital role in the function of neuroendocrine and cardiovascular systems. In this article, we report on the molecular and functional basis of alpha(1D) Ca(2+) channel modulation by protein kinase C (PKC). Specifically, we show that the serine 81 (S81) phosphorylation site at the NH(2)-terminal region plays a critical role in alpha(1D) Ca(2+) channel modulation by PKC. The introduction of a negatively charged residue at position 81, by converting serine to aspartate, mimicked the PKC phosphorylation effect on alpha(1D) Ca(2+) channel. The modulation of alpha(1D) Ca(2+) channel by PKC was prevented by dialyzing cells with a 35-amino acid peptide mimicking the alpha(1D) NH(2)-terminal region comprising S81. In addition, the data revealed that only betaII- and epsilonPKC isozymes are implicated in this regulation. These novel findings have significant implications in the pathophysiology of alpha(1D) Ca(2+) channel and in the development of PKC isozyme-targeted therapeutics.

Novel molecular mechanism involving alpha1D (Cav1.3) L-type calcium channel in autoimmune-associated sinus bradycardia.

BACKGROUND: Congenital heart block (CHB) is an autoimmune disease that affects fetuses/infants born to mothers with anti-Ro/La antibodies (positive IgG). Although the hallmark of CHB is complete atrioventricular block, sinus bradycardia has been reported recently in animal models of CHB. Interestingly, knockout of the neuroendocrine alpha1D Ca channel in mice results in significant sinus bradycardia and atrioventricular block, a phenotype reminiscent to that seen in CHB. Here, we tested the hypothesis that the alpha1D Ca channel is a novel target for positive IgG. METHODS AND RESULTS: Reverse transcription-polymerase chain reaction, confocal indirect immunostaining, and Western blot data established the expression of the alpha1D Ca channel in the human fetal heart. The effect of positive IgG on alpha1D Ca current (I(Ca-L)) was characterized in heterologous expression systems (tsA201 cells and Xenopus oocytes) because of the unavailability of alpha1D-specific modulators. alpha1D I(Ca-L) activated at negative potentials (between -60 and -50 mV). Positive IgG inhibited alpha1D I(Ca-L) in both expression systems. This inhibition was rescued by a Ca channel activator, Bay K8644. No effect on alpha1D I(Ca-L) was observed with negative IgG and denatured positive IgG. Western blot data showed that positive IgG binds directly to alpha1D Ca channel protein. CONCLUSIONS: The data are the first to demonstrate (1) expression of the alpha1D Ca channel in human fetal heart, (2) inhibition of alpha1D I(Ca-L) by positive IgG, and (3) direct cross-reactivity of positive IgG with the alpha1D Ca channel protein. Given that alpha1D I(Ca-L) activates at voltages within the pacemaker's diastolic depolarization, inhibition of alpha1D I(Ca-L) in part may account for autoimmune-associated sinus bradycardia. In addition, Bay K8644 rescue of alpha1D I(Ca-L) inhibition opens new directions in the development of pharmacotherapeutic approaches in the management of CHB.

Localization and modulation of {alpha}1D (Cav1.3) L-type Ca channel by protein kinase A.

Alpha1D L-type Ca channel was assumed to be of neuroendocrine origin only; however, alpha1D L-type Ca channel knockout mice exhibit sinus bradycardia and atrioventricular block, indicating a distinct role of alpha1D in the heart. The presence and distribution of alpha1D Ca channel in the heart and its regulation by protein kinase A (PKA) are just emerging. Our objective was to examine the localization of alpha1D L-type Ca channel in rabbit and rat hearts and its modulation by PKA. Here, we show the exclusive presence of alpha1D Ca channel transcript in the sinoatrial node, atrioventricular node, and atria but not in the ventricle by RT-PCR and the expression of alpha1D Ca channel protein in atrial myocytes' sarcolemma by indirect immunostaining and Western blot. There is no significant difference in the expression level of alpha1D Ca channel in the left versus right atrium. Superfusion of membrane-permeable 8-bromo-cAMP resulted in a significant increase of the peak current density of alpha1D Ca current expressed in tsA201 cells. This increase was inhibited by the PKA inhibitor (PKI). Application of 8-bromo-cAMP also readily phosphorylated the alpha1D Ca channel protein. The results are first to demonstrate that PKA phosphorylation of L-type Ca channel alpha1D-subunit resulted in an increase of the alpha1D Ca channel activity. Together with the observation that alpha1D Ca channel is exclusively present in the sinoatrial node and atria, the findings suggest that alpha1D Ca channel plays a unique role in the sinoatrial tissue and is a target for sympathetic control of heart rhythm.

Loss of function associated with novel mutations of the SCN5A gene in patients with Brugada syndrome.

BACKGROUND: Ventricular fibrillation is one of the leading causes of death in North America. Brugada syndrome is characterized by ST segment elevation on the right precordial leads V1 through V3 and right bundle branch block, and may cause sudden death. Mutations in the SCN5A gene encoding the cardiac voltage-gated Na+ channel (hNav1.5) are associated with Brugada syndrome. OBJECTIVES: In this study, three novel mutations on the SCN5A gene were identified and characterized in different patients with Brugada syndrome. METHODS: Blood samples were collected from patients with Brugada syndrome for gene screening. Mutations found on the SCN5A gene in these patients were reproduced in vitro on hNav1.5 background. Wild type and mutant channels expressed in tsA201 cells were characterized using the patch clamp technique in whole cell configuration and/or confocal microscopy. RESULTS: No current could be recorded from cells expressing the hNav1.5/G1740R mutant, incubated at 37 degrees C. However, at a lower incubation temperature (22 degrees C), macroscopic Na+ currents were recorded. Confocal microscopy study confirmed that at 37 degrees C, hNav1.5/G1740R mutant channels were retained in the endoplasmic reticulum. The E473X and N1774+12X mutants produced truncated proteins and did not express any currents; however, coexpression of each of these mutants with wild type channels shows 50% reduction of Na+ currents. CONCLUSION: This study confirms that the loss of function of cardiac Na+ channels is the basis of the Brugada syndrome clinical phenotype.

A newly characterized SCN5A mutation underlying Brugada syndrome unmasked by hyperthermia.

Febrile illness has been rarely reported to modulate ST segment elevation in right precordial leads on ECG or even precipitate ventricular fibrillation in patients with Brugada syndrome. We report the case of a patient whose Brugada ECG pattern was unmasked by hyperthermia secondary to acute cholangitis. Serial ECGs showed progressive attenuation of ST segment elevation as body temperature gradually returned to normal. Structural heart disease was ruled out. Intravenous flecainide injection reproduced a less remarkable ST segment elevation. Genetic screening demonstrated a single amino acid substitution (H681P) in the SCN5A gene, thus confirming the diagnosis of Brugada syndrome. In vitro expression of this newly characterized genetic defect revealed novel biophysical abnormalities consisting of a shift in both steady-state activation and inactivation, resulting in a 60% reduction of sodium window current. Thus, SCN5A-H681P mutation induces a significant loss of transmembrane current and is clinically associated with a pathologic phenotype that is elicited by hyperthermia. Overall the observed clinical features are in agreement with previous observations and strongly suggest that fever may be an environmental modifier among Brugada syndrome patients with a detrimental (and possibly arrhythmogenic) effect on cardiac repolarization.

Expression and intracellular localization of an SCN5A double mutant R1232W/T1620M implicated in Brugada syndrome.

Brugada syndrome is an inherited cardiac disorder caused by mutations in the cardiac sodium channel gene, SCN5A, that leads to ventricular fibrillation and sudden death. This study reports the changes in functional expression and cellular localization of an SCN5A double mutant (R1232W/T1620M) recently discovered in patients with Brugada syndrome. Mutant and wild-type (WT) human heart sodium channels (hNa(v)1.5) were expressed in tsA201 cells in the presence of the beta(1)-auxiliary subunit. Patch-clamp experiments in whole-cell configuration were conducted to assess functional expression. Immunohistochemistry and confocal microscopy were used to determine the spatial distribution of either WT or mutant cardiac sodium channels. The results show an abolition of functional sodium channel expression of the hNa(v)1.5/R1232W/T1620M mutant in the tsA201 cells. A conservative positively charged mutant, hNa(v)1.5/R1232K/T1620M, produced functional channels. Immunofluorescent staining showed that the FLAG-tagged hNa(v)1.5/WT transfected into tsA201 cells was localized on the cell surface, whereas the FLAG-tagged hNa(v)1.5/R1232W/T1620M mutant was colocalized with calnexin within the endoplasmic reticulum (ER). These results indicate that a positively charged arginine or lysine residue at position 1232 in the double mutant is required for the proper transport and functional expression of the hNa(v)1.5 protein. These results support the concept that loss of function of the cardiac Na(+) channel is responsible for the Brugada syndrome. The full text of this article is available at http://www.circresaha.org.