Impaired Ca2+ homeostasis is associated with atrial fibrillation in the alpha1D L-type Ca2+ channel KO mouse.
ABSTRACT The novel alpha1D Ca2+ channel together with alpha1C Ca2+ channel contribute to the L-type Ca2+ current (I(Ca-L)) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the alpha1D gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the alpha1D Ca2+ channel to the global I(Ca-L), the intracellular Ca2+ transient, the Ca2+ handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from alpha1D 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 alpha1D KO atrial myocytes revealed a significant reduction of I(Ca-L) (24.5%; P < 0.05). However, there were no changes in other currents such as I(Na), I(Ca-T), I(K), I(f), and I(to) and no changes in alpha1C mRNA levels of alpha1D KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular Ca2+ transient (approximately 40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR Ca2+ content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR Ca2+ release in alpha1D KO atria. In conclusion, genetic deletion of alpha1D Ca2+ channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the alpha1D KO mice were associated with a decrease in the total I(Ca-L) density, a reduction in intracellular Ca2+ transient, and impaired intracellular Ca2+ handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the alpha1D KO mice.
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ABSTRACT: The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.The Journal of Cell Biology 06/1995; 129(3):673-82. · 10.82 Impact Factor
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ABSTRACT: In cochlea inner hair cells (IHCs), L-type Ca(2+) channels (LTCCs) formed by alpha1D subunits (D-LTCCs) possess biophysical and pharmacological properties distinct from those of alpha1C containing C-LTCCs. We investigated to which extent these differences are determined by alpha1D itself by analyzing the biophysical and pharmacological properties of cloned human alpha1D splice variants in tsA-201 cells. Variant alpha1D(8A,) containing exon 8A sequence in repeat I, yielded alpha1D protein and L-type currents, whereas no intact protein and currents were observed after expression with exon 8B. In whole cell patch-clamp recordings (charge carrier 15-20 mm Ba(2+)), alpha1D(8A) - mediated currents activated at more negative voltages (activation threshold, -45.7 versus -31.5 mV, p < 0.05) and more rapidly (tau(act) for maximal inward currents 0.8 versus 2.3 ms; p < 0.05) than currents mediated by rabbit alpha1C. Inactivation during depolarizing pulses was slower than for alpha1C (current inactivation after 5-s depolarizations by 90 versus 99%, p < 0.05) but faster than for LTCCs in IHCs. The sensitivity for the dihydropyridine (DHP) L-type channel blocker isradipine was 8.5-fold lower than for alpha1C. Radioligand binding experiments revealed that this was not due to a lower affinity for the DHP binding pocket, suggesting that differences in the voltage-dependence of DHP block account for decreased sensitivity of D-LTCCs. Our experiments show that alpha1D(8A) subunits can form slowly inactivating LTCCs activating at more negative voltages than alpha1C. These properties should allow D-LTCCs to control physiological processes, such as diastolic depolarization in sinoatrial node cells, neurotransmitter release in IHCs and neuronal excitability.Journal of Biological Chemistry 06/2001; 276(25):22100-6. · 4.65 Impact Factor
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ABSTRACT: We have previously shown that alpha1-adrenergic activation inhibited beta-adrenergic-stimulated L-type Ca2+ current (I(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4beta-phorbol 12-myristate 13-acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 micromol/L) consistently inhibited basal I(Ba) by 40.5+/-7.4% and isoproterenol (ISO, 0.1 micromol/L)-stimulated I(Ba) by 48.9+/-7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue alpha-phorbol 12,13-didecanoate (0.1 micromol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (betaC2-2 and betaC2-4) specifically inhibit the translocation and function of C2-containing isozymes (alpha-PKC, betaI-PKC, and betaII-PKC), but not the C2-less isozymes (delta-PKC and epsilon-PKC). We first used the pseudosubstrate peptide (0.1 micromol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ba) was reduced to 16.8+/-7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2-derived peptides or control peptides. When the pipette contained 0.1 micromol/L of betaC2-2 or betaC2-4, PMA-induced inhibition of basal I(Ba) was 26.1+/-4.5% and 23.6+/-2.2%, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9+/-6.6% and 29.3+/-7.8% in the presence of betaC2-2 and betaC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled betaC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both betaC2-2 and betaC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.Circulation Research 06/1997; 80(5):720-9. · 11.86 Impact Factor