Irene Adler - Academia.edu (original) (raw)
Papers by Irene Adler
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles origi... more Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at:
Ectopic expression of the sarcoplasmic reticulum (SR) Ca 21 ATPase (SERCA) 1a pump in the mouse h... more Ectopic expression of the sarcoplasmic reticulum (SR) Ca 21 ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca 21 transients; however, the cellular mechanisms underlying altered Ca 21 handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca 21 load measurements revealed that TG myocytes have significantly enhanced SR Ca 21 lo d. Confocal line-scan images of field-stimulated SR Ca release showed an increased rate of Ca 21 removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by '30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca 21 current density was reduce...
![Research paper thumbnail of [Ca2+]ihomeostasis and cyclic nucleotide relaxation in aorta of phospholamban-deficient mice](https://attachments.academia-assets.com/105278522/thumbnails/1.jpg)
](American Journal of Physiology-Heart and Circulatory Physiology, 1999
Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+... more Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+-ATPase; phosphorylation of PLB relieves this inhibition. We previously reported significant differences in contractility in aorta from mice in which the gene for PLB was ablated (PLB−). In this study, we measured intracellular Ca2+concentration ([Ca2+]i) with fura 2 in the intact mouse aorta to more directly test the hypothesis that these changes are ascribable to altered SR function in vivo. Ten micromoles per liter of the α-agonist phenylephrine (PE) increased [Ca2+]imonotonically to a steady state in the wild-type aorta. In contrast, in PLB− aorta there was an initial rapid increase to a peak [Ca2+]i, which then decreased to a steady state that was lower than that in the wild type. Upon removal of the stimulus (either PE or KCl), the decrease in [Ca2+]iwas two times as fast in the PLB− as in the wild-type aorta. There were no significant differences between PLB− and wild-type aortas ...
Developments in Cardiovascular Medicine, 2001
The last few years have seen an explosion in the number of transgenic mouse models being generate... more The last few years have seen an explosion in the number of transgenic mouse models being generated. Exciting new data have been obtained. In particular, in the field of cardiac electrophysiology, the use of single-cell patch-clamp techniques on isolated cardiomyocytes offers a powerful method with which we can utilize to analyze the effects of selective genetic manipulation on the electrophysiological process of the cells.
American journal of physiology. Heart and circulatory physiology, 2003
Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardio... more Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardiomyopathy. Clinical treatment using beta-adrenergic receptor antagonists (beta-blockers) slows deterioration of cardiac function in end-stage heart failure patients; however, the effects of beta-blocker treatment on Ca2+ dynamics in the failing heart are unknown. To address this issue, tropomodulin-overexpressing transgenic (TOT) mice, which suffer from dilated cardiomyopathy, were treated with a nonselective beta-receptor blocker (5 mg. kg-1. day-1 propranolol) for 2 wk. Ca2+ dynamics in isolated cardiomyocytes of TOT mice significantly improved after treatment compared with untreated TOT mice. Frequency-dependent diastolic and Ca2+ transient amplitudes were returned to normal in propranolol-treated TOT mice and but not in untreated TOT mice. Ca2+ kinetic measurements of time to peak and time decay of the caffeine-induced Ca2+ transient to 50% relaxation were also normalized. Immunoblot ...
Journal of Molecular and Cellular Cardiology, 2001
Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN)... more Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN) mice produces a profound hypertrophy that rapidly progresses to heart failure. While calcineurin is regulated by Ca 2+ , the potential effects of calcineurin on cardiac myocyte Ca 2+ handling has not been evaluated. To this end, we examined L-type Ca 2+ currents (I Ca) in left ventricular myocytes. CLN myocytes had larger (≈80%) cell capacitance and enhanced I Ca density (≈20%) compared with non-transgenic (NTG) littermates, but no change in the current-voltage relationship, single-channel conductance or protein levels of 1 or 2 subunit of L-type Ca 2+ channels. Interestingly, the kinetics of I Ca inactivation was faster (≈two-fold) in CLN myocytes compared with NTG myocytes. Ryanodine application slowed the rate of I Ca inactivation in both groups and abolished the kinetic difference, suggesting that Ca 2+-dependent inactivation is increased in CLN myocytes due to altered SR Ca 2+ release. Treatment of CLN mice with Cyclosporine A (CsA), a calcineurin inhibitor, prevented myocyte hypertrophy and changes in I Ca activity and inactivation kinetics. However, there was no direct effect of CsA on I Ca in either NTG or CLN myocytes, suggesting that endogenous calcineurin activity does not directly regulate Ca 2+ channel activity. This interpretation is consistent with the observation that I Ca density, inactivation kinetics and regulation by isoproterenol were normal in cardiac-specific transgenic mice expressing calcineurin inhibitory protein domains from either Cain or AKAP79. Taken together these data suggest that chronic activation of calcineurin is associated with myocyte hypertrophy and a secondary enhancement of intracellular Ca 2+ handling that is tied to the hypertrophy response itself.
Journal of Molecular and Cellular Cardiology, 2000
Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; howeve... more Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; however, the exact mechanisms responsible for altered gene expression are poorly defined. The goal of this study was to investigate whether signaling kinases that are activated during cardiac hypertrophy directly modulate transcription factor activity and regulate gene expression. In an effort to understand this process, we focused our studies on the transcriptional activation of c-fos gene through the serum response element (SRE)/ternary complex factor (TCF) element, during phenylephrine-induced myocyte hypertrophy. In this study, we show that phosphorylated Elk-1, a TCF, binds to c-fos SRE and its binding to SRE is increased upon phenylephrine stimulation. Phenylephrine treatment activates phosphorylation of Elk-1 in the nucleus within five minutes and Elk-1-dependent transcriptional activation is abolished by inhibitors selective for MEK/ ERK kinases. These studies implicate that phosphorylation of Elk-1 by ERK kinase pathway is important for early gene activation during phenylephrine-induced myocyte hypertrophy.
Journal of Biological Chemistry, 2000
A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉-ATPas... more A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by ϳ35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca 2؉ homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca 2؉ stores and Ca 2؉ release in myocytes of SERCA2 heterozygous mice were decreased by ϳ40-60% and ϳ30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by ϳ40%. However, the rate of Ca 2؉ transient decline () was not altered significantly, suggesting that compensation was occurring in the removal of Ca 2؉ from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by ϳ40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased ϳ2and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na ؉-Ca 2؉ exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca 2؉ homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na ؉-Ca 2؉ exchanger.
Journal of Biological Chemistry, 2000
The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 g... more The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca 2؉ affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately ϳ20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calciumdependent calcium uptake measurements showed that the maximal velocity of Ca 2؉ uptake was not changed, but the apparent pump affinity for Ca 2؉ (K 0.5) was increased in SERCA2b transgenic mice (0.199 ؎ 0.011 M) compared with wild-type control mice (0.269 ؎ 0.012 M, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.
Circulation Research, 2001
Ectopic expression of the sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) 1a pump in the mouse h... more Ectopic expression of the sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca 2+ transients; however, the cellular mechanisms underlying altered Ca 2+ handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca 2+ load measurements revealed that TG myocytes have significantly enhanced SR Ca 2+ load. Confocal line-scan images of field-stimulated SR Ca 2+ release showed an increased rate of Ca 2+ removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by ≈30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca 2+ current density was reduced...
Circulation Research, 1997
Phospholamban (PLB) is a protein associated with the Ca 2+ -ATPase of the sarcoplasmic reticulum ... more Phospholamban (PLB) is a protein associated with the Ca 2+ -ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca 2+ -ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB − ). The PLB − mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB − aorta, suggesting changes in SR Ca 2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain ...
Pfl�gers Archiv European Journal of Physiology, 1998
Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the my... more Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the myocyte to its electrical activity via a mechanism that is not well understood. Recent pharmacological evidence suggests that KATP channels may mediate ischemic preconditioning. However, there is no potent pharmaceutical agent that specifically blocks the sarcolemmal KATP channel without significant effects on other cellular proteins. As a molecular tool, the GFG sequence in the H5 loop of the murine Kir6.2 channel was mutated to AFA. This mutated channel subunit (6.2AFA) suppressed wild-type Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, whole-cell KATP current recorded from HEK cells was greatly attenuated. The 6.2AFA subunit also co-assembled with endogenous subunits in both smooth-muscle-derived A10 cells and rat neonatal ventricular myocytes, resulting in a significant reduction of current compared with that recorded from non-transfected or mock-transfected cells (<15% of control for both cell types). This study shows that mutation of GFG-->AFA in the putative pore-forming region of Kir6.2 acts in a dominant-negative manner to suppress current in heterologous systems and in native cells.
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles origi... more Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at:
Ectopic expression of the sarcoplasmic reticulum (SR) Ca 21 ATPase (SERCA) 1a pump in the mouse h... more Ectopic expression of the sarcoplasmic reticulum (SR) Ca 21 ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca 21 transients; however, the cellular mechanisms underlying altered Ca 21 handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca 21 load measurements revealed that TG myocytes have significantly enhanced SR Ca 21 lo d. Confocal line-scan images of field-stimulated SR Ca release showed an increased rate of Ca 21 removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by '30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca 21 current density was reduce...
American Journal of Physiology-Heart and Circulatory Physiology, 1999
Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+... more Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+-ATPase; phosphorylation of PLB relieves this inhibition. We previously reported significant differences in contractility in aorta from mice in which the gene for PLB was ablated (PLB−). In this study, we measured intracellular Ca2+concentration ([Ca2+]i) with fura 2 in the intact mouse aorta to more directly test the hypothesis that these changes are ascribable to altered SR function in vivo. Ten micromoles per liter of the α-agonist phenylephrine (PE) increased [Ca2+]imonotonically to a steady state in the wild-type aorta. In contrast, in PLB− aorta there was an initial rapid increase to a peak [Ca2+]i, which then decreased to a steady state that was lower than that in the wild type. Upon removal of the stimulus (either PE or KCl), the decrease in [Ca2+]iwas two times as fast in the PLB− as in the wild-type aorta. There were no significant differences between PLB− and wild-type aortas ...
Developments in Cardiovascular Medicine, 2001
The last few years have seen an explosion in the number of transgenic mouse models being generate... more The last few years have seen an explosion in the number of transgenic mouse models being generated. Exciting new data have been obtained. In particular, in the field of cardiac electrophysiology, the use of single-cell patch-clamp techniques on isolated cardiomyocytes offers a powerful method with which we can utilize to analyze the effects of selective genetic manipulation on the electrophysiological process of the cells.
American journal of physiology. Heart and circulatory physiology, 2003
Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardio... more Changes in calcium (Ca2+) regulation contribute to loss of contractile function in dilated cardiomyopathy. Clinical treatment using beta-adrenergic receptor antagonists (beta-blockers) slows deterioration of cardiac function in end-stage heart failure patients; however, the effects of beta-blocker treatment on Ca2+ dynamics in the failing heart are unknown. To address this issue, tropomodulin-overexpressing transgenic (TOT) mice, which suffer from dilated cardiomyopathy, were treated with a nonselective beta-receptor blocker (5 mg. kg-1. day-1 propranolol) for 2 wk. Ca2+ dynamics in isolated cardiomyocytes of TOT mice significantly improved after treatment compared with untreated TOT mice. Frequency-dependent diastolic and Ca2+ transient amplitudes were returned to normal in propranolol-treated TOT mice and but not in untreated TOT mice. Ca2+ kinetic measurements of time to peak and time decay of the caffeine-induced Ca2+ transient to 50% relaxation were also normalized. Immunoblot ...
Journal of Molecular and Cellular Cardiology, 2001
Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN)... more Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN) mice produces a profound hypertrophy that rapidly progresses to heart failure. While calcineurin is regulated by Ca 2+ , the potential effects of calcineurin on cardiac myocyte Ca 2+ handling has not been evaluated. To this end, we examined L-type Ca 2+ currents (I Ca) in left ventricular myocytes. CLN myocytes had larger (≈80%) cell capacitance and enhanced I Ca density (≈20%) compared with non-transgenic (NTG) littermates, but no change in the current-voltage relationship, single-channel conductance or protein levels of 1 or 2 subunit of L-type Ca 2+ channels. Interestingly, the kinetics of I Ca inactivation was faster (≈two-fold) in CLN myocytes compared with NTG myocytes. Ryanodine application slowed the rate of I Ca inactivation in both groups and abolished the kinetic difference, suggesting that Ca 2+-dependent inactivation is increased in CLN myocytes due to altered SR Ca 2+ release. Treatment of CLN mice with Cyclosporine A (CsA), a calcineurin inhibitor, prevented myocyte hypertrophy and changes in I Ca activity and inactivation kinetics. However, there was no direct effect of CsA on I Ca in either NTG or CLN myocytes, suggesting that endogenous calcineurin activity does not directly regulate Ca 2+ channel activity. This interpretation is consistent with the observation that I Ca density, inactivation kinetics and regulation by isoproterenol were normal in cardiac-specific transgenic mice expressing calcineurin inhibitory protein domains from either Cain or AKAP79. Taken together these data suggest that chronic activation of calcineurin is associated with myocyte hypertrophy and a secondary enhancement of intracellular Ca 2+ handling that is tied to the hypertrophy response itself.
Journal of Molecular and Cellular Cardiology, 2000
Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; howeve... more Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; however, the exact mechanisms responsible for altered gene expression are poorly defined. The goal of this study was to investigate whether signaling kinases that are activated during cardiac hypertrophy directly modulate transcription factor activity and regulate gene expression. In an effort to understand this process, we focused our studies on the transcriptional activation of c-fos gene through the serum response element (SRE)/ternary complex factor (TCF) element, during phenylephrine-induced myocyte hypertrophy. In this study, we show that phosphorylated Elk-1, a TCF, binds to c-fos SRE and its binding to SRE is increased upon phenylephrine stimulation. Phenylephrine treatment activates phosphorylation of Elk-1 in the nucleus within five minutes and Elk-1-dependent transcriptional activation is abolished by inhibitors selective for MEK/ ERK kinases. These studies implicate that phosphorylation of Elk-1 by ERK kinase pathway is important for early gene activation during phenylephrine-induced myocyte hypertrophy.
Journal of Biological Chemistry, 2000
A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉-ATPas... more A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by ϳ35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca 2؉ homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca 2؉ stores and Ca 2؉ release in myocytes of SERCA2 heterozygous mice were decreased by ϳ40-60% and ϳ30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by ϳ40%. However, the rate of Ca 2؉ transient decline () was not altered significantly, suggesting that compensation was occurring in the removal of Ca 2؉ from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by ϳ40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased ϳ2and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na ؉-Ca 2؉ exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca 2؉ homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na ؉-Ca 2؉ exchanger.
Journal of Biological Chemistry, 2000
The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 g... more The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca 2؉ affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately ϳ20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calciumdependent calcium uptake measurements showed that the maximal velocity of Ca 2؉ uptake was not changed, but the apparent pump affinity for Ca 2؉ (K 0.5) was increased in SERCA2b transgenic mice (0.199 ؎ 0.011 M) compared with wild-type control mice (0.269 ؎ 0.012 M, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.
Circulation Research, 2001
Ectopic expression of the sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) 1a pump in the mouse h... more Ectopic expression of the sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca 2+ transients; however, the cellular mechanisms underlying altered Ca 2+ handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca 2+ load measurements revealed that TG myocytes have significantly enhanced SR Ca 2+ load. Confocal line-scan images of field-stimulated SR Ca 2+ release showed an increased rate of Ca 2+ removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by ≈30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca 2+ current density was reduced...
Circulation Research, 1997
Phospholamban (PLB) is a protein associated with the Ca 2+ -ATPase of the sarcoplasmic reticulum ... more Phospholamban (PLB) is a protein associated with the Ca 2+ -ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca 2+ -ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB − ). The PLB − mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB − aorta, suggesting changes in SR Ca 2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain ...
Pfl�gers Archiv European Journal of Physiology, 1998
Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the my... more Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the myocyte to its electrical activity via a mechanism that is not well understood. Recent pharmacological evidence suggests that KATP channels may mediate ischemic preconditioning. However, there is no potent pharmaceutical agent that specifically blocks the sarcolemmal KATP channel without significant effects on other cellular proteins. As a molecular tool, the GFG sequence in the H5 loop of the murine Kir6.2 channel was mutated to AFA. This mutated channel subunit (6.2AFA) suppressed wild-type Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, whole-cell KATP current recorded from HEK cells was greatly attenuated. The 6.2AFA subunit also co-assembled with endogenous subunits in both smooth-muscle-derived A10 cells and rat neonatal ventricular myocytes, resulting in a significant reduction of current compared with that recorded from non-transfected or mock-transfected cells (<15% of control for both cell types). This study shows that mutation of GFG-->AFA in the putative pore-forming region of Kir6.2 acts in a dominant-negative manner to suppress current in heterologous systems and in native cells.