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The novel [[alpha].sub.1D] [Ca.sup.2+] channel together with [[alpha].sub.1C] [Ca.sup.2+] channel contribute to the L-type [Ca.sup.2+] current ([I.sub.Ca-L]) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the [[alpha].sub.1D] gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the [[alpha].sub.1D] [Ca.sup.2+] channel to the global [I.sub.Ca-L], the intracellular [Ca.sup.2+] transient, the [Ca.sup.2+] handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from [[alpha].sub.1D] KO mice demonstrated significant sinus bradycardia, atrioventricular block, and vulnerability to AF. The wild-type mice showed no ECG abnormalities and no AF. Patch-clamp recordings from isolated [[alpha].sub.1D] KO atrial myocytes revealed a significant reduction of [I.sub.Ca-L] (24.5%; P < 0.05). However, there were no changes in other currents such as [I.sub.Na], [I.sub.Ca-T], [I.sub.K], [I.sub.f], and [I.sub.to] and no changes in [[alpha].sub.1C] mRNA levels of [[alpha].sub.1D] KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular [Ca.sup.2+] transient (~40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR [Ca.sup.2+] content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR [Ca.sup.2+] release in am KO atria. In conclusion, genetic deletion of [[alpha].sub.1D] [Ca.sup.2+] channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the [[alpha].sub.1D] KO mice were associated with a decrease in the total [I.sub.Ca-L] density, a reduction in intracellular [Ca.sup.2+] transient, and impaired intracellular [Ca.sup.2+] handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the [[alpha].sub.1D] KO mice. mice; calcium transient; arrhythmia; myocyte

[[alpha].sub.1D] L-type Ca channel was assumed to be of neuroendocrine origin only: however, [[alpha].sub.1D] L-type Ca channel knockout mice exhibit sinus bradycardia and atrioventricular block, indicating a distinct role of [[alpha].sub.1D] in the heart. The presence and distribution of [[alpha].sub.1D] Ca channel in the heart and its regulation by protein kinase A (PKA) are just emerging. Our objective was to examine the localization of [[alpha].sub.1D] L-type Ca channel in rabbit and rat hearts and its modulation by PKA. Here, we show the exclusive presence of [[alpha].sub.1D] Ca channel transcript in the sinoatrial node, atrio-ventricular node, and atria but not in the ventricle by RT-PCR and the expression of [[alpha].sub.1D] Ca channel protein in atrial myocytes' sarcolemma by indirect immunostaining and Western blot. There is no significant difference in the expression level of [[alpha].sub.1D] 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 [[alpha].sub.1D] Ca current expressed in tsA201 cells. This increase was inhibited by the PKA inhibitor (PKI). Application of 8-bromo-cAMP also readily phosphorylated the [[alpha].sub.1D] Ca channel protein. The results are first to demonstrate that PKA phosphorylation of L-type Ca channel [[alpha].sub.1D]-subunit resulted in an increase of the [[alpha].sub.1D] Ca channel activity. Together with the observation that [[alpha].sub.1D] Ca channel is exclusively present in the sinoatrial node and atria, the findings suggest that [[alpha].sub.1D] Ca channel plays a unique role in the sinoatrial tissue and is a target for sympathetic control of heart rhythm. phosphorylation; protein kinase A; sinoatrial node

The role of mitochondrial free radicals in the cardioprotective effect of ischemic preconditioning was examined in isolated buffer-perfused rat hearts. Infarct size in control rat hearts subjected to 30 min of regional ischemia and 120 rain of reperfusion was 32.6 [+ or -] 3.4% of the risk zone. Ischemic preconditioning (3 cycles of 5-min global ischemia/5-min reperfusion) before the same regional ischemia and reperfusion protocol significantly reduced infarct size to 2.6 [+ or -] 0.8% of the risk zone. Perfusion with menadione (3.0 [micro]M), a generator of mitochondrial free radicals, in lieu of preconditioning ischemia significantly reduced infarction to 10.9 [+ or -] 2.7%. N-2-mercaptopropionylglycine (1.0 mM), a free radical scavenger, blocked the protection of menadione, significantly increasing infarction to 23.5 [+ or -] 1.1%. Myxothiazol (0.6 [micro]M), a site III mitochondrial inhibitor, blocked the protection of menadione and significantly increased infarction to 25.2 [+ or -] 3.8%. The infarct-limiting effect of menadione was attenuated to 19.7 [+ or -] 1.5% of the risk zone by 10 [micro]M SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Furthermore, menadione significantly increased p38 MAPK phosphorylation to a level 5.6-fold over basal. These results indicate that free radicals that originate within mitochondria can activate p38 MAPK and protect hearts against infarction. myocardial infarction; ischemia; mitochondria; p38 MAPK

Mancarella S, Yue Y, Karnabi E, Qu Y, El-Sherif N, Boutjdir M. Impaired Ca[sup2+] homeostasis is associated with atrial fibrillation in the α1D L-type Ca[sup2+] channel KU mouse. Am J Physiol Heart Circ Physiol 295: H2017-H2024, 2008. First published September 12, 2008; doi: 10.1152/ajpheart.00537.2008.-The novel α1D Ca[sup2+] channel together with α1D Ca[sup2+] channel contribute to the L-type Ca[sup2+] current (I[subCa-L]) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the α1D gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the α1D Ca[sup2+] channel to the global I[subCa-L], the intracellular Ca[sup2+] transient, the Ca[sup2+] handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from α1D KO mice demonstrated significant sinus bradycardia, atrioventricular block, and vulnerability to AF. The wild-type mice showed no ECG abnormalities and no AF. Patch-clamp recordings from isolated α1D KO atrial myocytes revealed a significant reduction of I[subCa-L] (24.5%; P < 0.05). However, there were no changes in other currents such as I[subNa], I[subCa-T], I[subK], If, and I[subf] and no changes in α[subIC] mRNA levels of α1D KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular Ca[sup2+] transient (-40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR Ca[sup2+] content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR Ca[sup2+] release in α1D KO atria. In conclusion, genetic deletion of α1D Ca[sup2+] channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the α1D KO mice were associated with a decrease in the total I[subCa-L] density, a reduction in intracellular Ca[sup2+] transient, and impaired intracellular Ca[sup2+] handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the α1D KO mice. [ABSTRACT FROM AUTHOR]

It is well established that β-adrenoceptor stimulation activates PKA and α1-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of α1-adrenoceptors inhibits β-adrenoceptor stimulated L-type Ca current (ICa-L) and direct activation of &epsiv;PKC leads to ICa-L inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of &epsiv;PKC alters ICa-L and its response to adrenergic stimulation. Therefore, we investigated the interaction between β- and α1-adrenoceptors vis-a&grave;-vis ICa-L in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of &epsiv;PKC (&epsiv;PKC agonist). Whole-cell ICa-L was recorded from &epsiv;PKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) β-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus α1-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of &epsiv;PKC leads to reduced basal ICa-L density. β-adrenoceptor activation of ICa-L is blunted in &epsiv;PKC agonist TG mice. α-adrenoceptor cross-talk with β-adrenoceptor signaling pathways vis-a&grave;-vis L-type Ca channels is impaired in &epsiv;PKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in &epsiv;PKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure. [Copyright &y& Elsevier]

Objectives: Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mKATP), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mKATP opening. In addition, we tested whether protection from anisomycin, a p38 MAPK activator, is dependent on ROS production. Methods and Results: Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC50 of 270 nM. Menadione’s infarct-limiting effect was insensitive to 200 μM 5-hydroxydecanoate (5HD), an mKATP channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger. Conclusions: These results indicate that mKATP opening occurs upstream of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent. [Copyright &y& Elsevier]

Abstract: Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have recently established that anti-SSA/Ro -SSB/La autoantibodies inhibit α1D L-type Ca current, ICa-L, and cross-react with the α1D Ca channel protein. This study aims at identifying the possible binding sites on α1D protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5–S6) of each of the four α1D Ca channel protein domains I–IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera containing anti-Ro/La autoantibodies from 118 mothers with CHB children and from 15 mothers with anti-Ro/La autoantibodies but have healthy children, and from 28 healthy mothers without anti-Ro/La autoantibodies and healthy children were evaluated. Seventeen of 118 (14.4%) sera from mothers with CHB children reacted with the extracellular loop of domain I S5–S6 region (E1). In contrast, only 2 of 28 (7%) of sera from healthy mothers (−anti-Ro/La) and healthy children reacted with E1 loop and none (0 of 15) of sera from healthy mothers (+anti-Ro/La) and healthy children reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D reading establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera and purified IgG showed inhibition (44.1% and 49.8%, respectively) of the α1D ICa-L expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 fusion protein. The results identified the extracellular loop of domain I S5–S6 of L-type Ca channel α1D subunit as a target for autoantibodies from a subset of mothers with CHB children. This novel finding provides insights into the potential development of therapeutic peptides that could bind to the pathogenic antibodies and prevent CHB. [Copyright &y& Elsevier]

It was recently reported that sera from patients with systemic lupus erythematosus contain antibodies reactive with the second extracellular loop of the serotoninergic 5-HT4 receptor expressed in the human heart. This antibody response was associated with antibodies to 52 kD SSA/Ro, a reactivity prevalent in mothers of children with congenital heart block (CHB). The current study was undertaken to determine whether the 5-HT4 receptor is a target of the immune response in these mothers. Initial experiments demonstrated mRNA expression of the 5-HT4 receptor in the human foetal atrium. Electrophysiologic studies established that human foetal atrial cells express functional 5-HT4 receptors. Sera from 116 mothers enrolled in the Research Registry for Neonatal Lupus, whose children have CHB, were evaluated. Ninety-nine (85%) of these maternal sera contained antibodies to SSA/Ro, 84% of which were reactive with the 52 kD SSA/Ro component by immunoblot. None of the 116 sera were reactive with the peptide spanning aa165–185 of the serotoninergic receptor. Rabbit antisera which recognized this peptide did not react with 52 kD SSA/Ro or peptide aa365–382 in the C terminus. Although 5-HT4 receptors are present and functional in the human foetal heart, maternal antibodies to the 5-HT4 receptor are not associated with the development of CHB. [Copyright &y& Elsevier]

Examines the ionic basis of the abnormal electrophysiological character of myocytes from the right ventricle of chronic hypoxic guinea pigs. Effect of chronic hypoxia; Role of attenuatement of the outward potassium in the prolongation of the action potential duration; Influence of metabolic changes on phospholyration and dephosphorylation.

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have recently established that anti-SSA/Ro -SSB/La autoantibodies inhibit alpha(1D) L-type Ca current, I(Ca-L), and cross-react with the alpha(1D) Ca channel protein. This study aims at identifying the possible binding sites on alpha(1D) protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four alpha(1D) Ca channel protein domains I-IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera containing anti-Ro/La autoantibodies from 118 mothers with CHB children and from 15 mothers with anti-Ro/La autoantibodies but have healthy children, and from 28 healthy mothers without anti-Ro/La autoantibodies and healthy children were evaluated. Seventeen of 118 (14.4%) sera from mothers with CHB children reacted with the extracellular loop of domain I S5-S6 region (E1). In contrast, only 2 of 28 (7%) of sera from healthy mothers (-anti-Ro/La) and healthy children reacted with E1 loop and none (0 of 15) of sera from healthy mothers (+anti-Ro/La) and healthy children reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D reading establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera and purified IgG showed inhibition (44.1% and 49.8%, respectively) of the alpha(1D) I(Ca-L) expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel alpha(1D) subunit as a target for autoantibodies from a subset of mothers with CHB children. This novel finding provides insights into the potential development of therapeutic peptides that could bind to the pathogenic antibodies and prevent CHB., (Published by Elsevier Ltd.)

It is well established that beta-adrenoceptor stimulation activates PKA and alpha(1)-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of alpha(1)-adrenoceptors inhibits beta-adrenoceptor stimulated L-type Ca current (I(Ca-L)) and direct activation of epsilonPKC leads to I(Ca-L) inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of epsilonPKC alters I(Ca-L) and its response to adrenergic stimulation. Therefore, we investigated the interaction between beta- and alpha(1)-adrenoceptors vis-à-vis I(Ca-L) in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of epsilonPKC (epsilonPKC agonist). Whole-cell I(Ca-L) was recorded from epsilonPKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) beta-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus alpha(1)-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of epsilonPKC leads to reduced basal I(Ca-L) density. beta-adrenoceptor activation of I(Ca-L) is blunted in epsilonPKC agonist TG mice. alpha-adrenoceptor cross-talk with beta-adrenoceptor signaling pathways vis-à-vis L-type Ca channels is impaired in epsilonPKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in epsilonPKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure.

This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries. On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer. The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

Objectives: Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mK(ATP)), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mK(ATP) opening. In addition, we tested whether protection from anisomycin, a p38 MAPK activator, is dependent on ROS production., Methods and Results: Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC(50) of 270 nM. Menadione's infarct-limiting effect was insensitive to 200 microM 5-hydroxydecanoate (5HD), an mK(ATP) channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger., Conclusions: These results indicate that mK(ATP) opening occurs upstream of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent.