F1000 - Post-publication peer review of the biomedical literature, 2012
Antagonists of L-type Ca 2+ channels (LTCCs) have been used to treat human cardiovascular disease... more Antagonists of L-type Ca 2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C-/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C-/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C-/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C +/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocytespecific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca 2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease. Conflict of interest: The authors have declared that no conflict of interest exists.
Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 2001
Inflammatory disturbances in the liver microcirculation have been associated with preservation in... more Inflammatory disturbances in the liver microcirculation have been associated with preservation injury of hepatic grafts. Vascular endothelial growth factor (VEGF), a proinflammatory growth factor released by hepatocytes, acts on sinusoidal endothelial cells, but its implication in graft injury is still unclear. We studied VEGF production by rat hepatocytes after cold ischemia and warm reoxygenation and compared the capacity of University of Wisconsin (UW) and sodium-lactobionate-sucrose (SLS) preservation solutions to maintain this hepatocellular function. Isolated hepatocytes were kept for 0, 24, and 48 hours at 4 degrees C in either solution (cold ischemia), then incubated for 1 to 24 hours at 37 degrees C (warm reoxygenation). We assessed cell viability and production of VEGF messenger RNA (mRNA) and protein. Cell viability decreased in a linear time-dependent fashion by 10% after 48 hours of cold preservation and by an additional 40% after 24 hours of warm culture. Very little V...
We have developed a mathematical model of the human atrial myocyte based on averaged voltage-clam... more We have developed a mathematical model of the human atrial myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley-type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action
19), P,0.001). Consistent with these findings were the results of the ribonuclease protection ass... more 19), P,0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding for IKur) in female compared with male ventricle. The additional K1 currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression
1. The properties of the slow inward 'tail currents' (I(tail)) that followed depo... more 1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.
Journal of Molecular and Cellular Cardiology, 1997
The radioligand [3H]dofetilide binds specifically to the delayed rectifier potassium channel and ... more The radioligand [3H]dofetilide binds specifically to the delayed rectifier potassium channel and provides a biochemical approach to study interactions of Class III drugs with this channel. However, previous studies have examined the binding of [3H]dofetilide to cardiac myocytes only at extracellular potassium of 135 mM. Because previous electrophysiological studies have shown that hyperkalemia could alter the pharmacological responses to I(Kr) channel blockers, the hypothesis tested in this study was that changing ionic conditions would alter characteristics of [3H]dofetilide binding. under physiological conditions (Na+ 135 mM, K+ 5 mM), [3H]dofetilide bound to two sites on guinea-pig ventricular myocytes (a high-affinity site, K(d) 26+/-8 nM, B(max) 81+/-12 fmol/10(6) cells: and a low-affinity site, K(d) 1.6+/-0.8 microM, B(max) 1003+/-173 fmol/10(6) cells, n=11). Binding properties were not altered by changes in osmolarity or extracellular sodium. However, when extracellular K+ was increased to 20 mM, a single binding site was observed with an affinity K(d) of 120+12 nM and a B(max) of 303+/-57 fmol/10(6) cells (P<0.05; n=6). To establish whether this effect was mediated at the high-affinity site we assessed the effects of elevated extracellular potassium on a biological model, neonatal mouse myocytes, that expressed solely the high-affinity binding sites. The K(d) values for binding to fetal mouse cardiac myocytes at an extracellular K+ of 5 mM and 20 mM were also significantly different, 29+/-10 and 230+/-46 nM, respectively. In conclusion, [3H]dofetilide binding to its high-affinity site is modulated by extracellular potassium.
p38 MAPK Isoforms mRNA Protein Heart Hypertrophy Chronic pressure overload p38 mitogen-activated ... more p38 MAPK Isoforms mRNA Protein Heart Hypertrophy Chronic pressure overload p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: α, β, γ, and δ. To date, the prominent isoform in heart has been thought to be p38α. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38γ and p38δ were expressed at protein levels comparable to p38α and 38β, respectively. In the early phase of pressure-overload hypertrophy (1-7 days after constriction of the transverse aorta), the abundance of p38β, p38γ and p38δ mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38α and p38γ in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7-28 days after constriction of the transverse aorta), p38γ immunoreactivity accumulated in the nucleus whereas the distribution of p38α remained unaffected. Hence, both p38α and p38γ are prominent p38 isoforms in heart and p38γ may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.
In most cases, the underlying cause of death is the rapid onset of lethal ventricular arrhythmias... more In most cases, the underlying cause of death is the rapid onset of lethal ventricular arrhythmias in the setting of acquired heart disease. The onset of sudden cardiac death is multifactorial, and the elucidation of the precise molecular and cellular pathways responsible for this phenotype has been difficult due to the paucity of ge-Robert G. Gourdie, 3 Marc M. Rahme, 2 netic insights into this complex human cardiac disease. ). However, studies of affected families have re-La Jolla, California 92093 vealed that only 20% of the patients who harbor the 3 Department of Cell Biology and Anatomy disease genotype display sudden death (Roden and Medical University of South Carolina Spooner, 1999), suggesting the requirement of addi-Charleston, South Carolina 29425 tional pathways that must be present to produce the 4 University of Calgary disease phenotype. Further evidence of the requirement Calgary T2N 4N1 of a "second hit" to produce lethal ventricular arrhyth-Canada mogenesis has come from studies of genetically engineered mice that harbor mutations in the cardiac Na ϩ or K ϩ channels that produce long QT syndrome, prolonged Summary cardiac repolarization, and associated arrhythmias, but not sudden cardiac death (Barry et al., 1998; Charpentier HF-1b, an SP1-related transcription factor, is preferenet al., 1998; Drici et al., 1998; London et al., 1998; Abbott tially expressed in the cardiac conduction system and et al., 1999; Kupershmidt et al., 1999). Currently, there ventricular myocytes in the heart. Mice deficient for
Sex differences in cardiac electrophysiological properties and arrhythmias are evident in epidemi... more Sex differences in cardiac electrophysiological properties and arrhythmias are evident in epidemiologic and investigative studies as well as in daily patient care. At the supraventricular level, women are at increased risk of sick sinus syndrome and atrioventricular (AV) node re-entrant tachycardia, whereas men manifest more AV block and accessory pathway-mediated arrhythmias. At the ventricular level, women are generally at higher risk of long QT-associated arrhythmias, whereas men are more likely to present with early repolarization, idiopathic ventricular fibrillation, and Brugada syndromes. Great advances have been made in unraveling the fundamental mechanisms underlying sex differences in ventricular arrhythmias, particularly those associated with abnormal repolarization. Conversely, the basis for male-predominant arrhythmia risk in structural heart disease and differences in supraventricular arrhythmia susceptibility are poorly understood. Beyond biological differences, arrhythmia occurrence and patient care decisions are also influenced by gender-related factors. This article reviews the current knowledge regarding the nature and underlying mechanisms of sex differences in basic cardiac electrophysiology and clinical arrhythmias.
In this study, we report diVerent protocols used to obtain highly enriched and well-characterized... more In this study, we report diVerent protocols used to obtain highly enriched and well-characterized protein fractions that could be used to determine the subcellular localization of proteins. DiVerent protein fractions (total, cytosolic, total membrane, sarcolemmal, and nuclear) were isolated from mouse heart by a combination of either polytron homogenization or liquid nitrogen pulverization followed by density gradient centrifugation. Triton X-100 was used in speciWc fractions to help in the solubilization of proteins obtained with fractionation protocols. Following the isolation, enzymatic assays and Western blot analysis were used to evaluate the enrichment and/or cross-contamination of these protein fractions. Glucose-6-phosphate dehydrogenase, Na + /K + -ATPase, mitochondrial Ca 2+ -ATPase, sarco-endoplasmic reticulum Ca 2+ -ATPase, glucose-regulated protein, and nucleoporin P62 were used as speciWc markers for the cytosol, sarcolemma, mitochondria, sarco-endoplasmic reticulum, endoplasmic reticulum, and nucleus, respectively. The results show that we obtained enriched protein fractions with little to no crosscontamination. These puriWcation protocols allow us to obtain diVerent protein fractions that could be used in a wide variety of studies.
MK5, a member of the MAPK-activated protein kinase family, is highly expressed in heart. Whereas ... more MK5, a member of the MAPK-activated protein kinase family, is highly expressed in heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart. mRNA for 5 splice variants (MK5.1-5.5), including the original form (MK5.1), was detected. MK5 comprises 14 exons: exon 12 splicing was modified in MK5.2, MK5.3, and MK5.5. MK5.2 and MK5.5 lacked 6 bases at the 3′-end of exon 12, whereas MK5.3 lacked exon 12, resulting in a frame-shift and premature termination of translation at codon 3 of exon 13. MK5.4 and MK5.5 lacked exons 2-6, encoding kinase subdomains IVI, and were kinase-dead. All 5 MK5 variants were detected at the mRNA level in all mouse tissues examined; however, their relative abundance was tissue-specific. Furthermore, the relative abundance of variant mRNA was altered both during hypertrophy and postnatal cardiac development, suggesting the generation or the stability of MK5 variant mRNAs is subject to regulation. When expressed in HEK293 cells, MK5.1, MK5.2 and MK5.3 were nuclear whereas MK5.4 and MK5.5 were cytoplasmic. A p38 MAPK activator, anisomycin, induced the redistribution of each variant. In contrast, MK5 co-immunoprecipitated ERK3, but not ERK4 or p38α, in control and hypertrophying hearts. GST pull-down assays revealed unbound ERK4 and p38α but no free MK5 or ERK3 in heart lysates. Hence, 1) in heart MK5 complexes with ERK3 and 2) MK5 splice variants may mediate distinct effects thus increasing the functional diversity of ERK3-MK5 signaling.
F1000 - Post-publication peer review of the biomedical literature, 2012
Antagonists of L-type Ca 2+ channels (LTCCs) have been used to treat human cardiovascular disease... more Antagonists of L-type Ca 2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C-/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C-/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C-/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C +/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocytespecific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca 2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease. Conflict of interest: The authors have declared that no conflict of interest exists.
Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 2001
Inflammatory disturbances in the liver microcirculation have been associated with preservation in... more Inflammatory disturbances in the liver microcirculation have been associated with preservation injury of hepatic grafts. Vascular endothelial growth factor (VEGF), a proinflammatory growth factor released by hepatocytes, acts on sinusoidal endothelial cells, but its implication in graft injury is still unclear. We studied VEGF production by rat hepatocytes after cold ischemia and warm reoxygenation and compared the capacity of University of Wisconsin (UW) and sodium-lactobionate-sucrose (SLS) preservation solutions to maintain this hepatocellular function. Isolated hepatocytes were kept for 0, 24, and 48 hours at 4 degrees C in either solution (cold ischemia), then incubated for 1 to 24 hours at 37 degrees C (warm reoxygenation). We assessed cell viability and production of VEGF messenger RNA (mRNA) and protein. Cell viability decreased in a linear time-dependent fashion by 10% after 48 hours of cold preservation and by an additional 40% after 24 hours of warm culture. Very little V...
We have developed a mathematical model of the human atrial myocyte based on averaged voltage-clam... more We have developed a mathematical model of the human atrial myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley-type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action
19), P,0.001). Consistent with these findings were the results of the ribonuclease protection ass... more 19), P,0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding for IKur) in female compared with male ventricle. The additional K1 currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression
1. The properties of the slow inward 'tail currents' (I(tail)) that followed depo... more 1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.
Journal of Molecular and Cellular Cardiology, 1997
The radioligand [3H]dofetilide binds specifically to the delayed rectifier potassium channel and ... more The radioligand [3H]dofetilide binds specifically to the delayed rectifier potassium channel and provides a biochemical approach to study interactions of Class III drugs with this channel. However, previous studies have examined the binding of [3H]dofetilide to cardiac myocytes only at extracellular potassium of 135 mM. Because previous electrophysiological studies have shown that hyperkalemia could alter the pharmacological responses to I(Kr) channel blockers, the hypothesis tested in this study was that changing ionic conditions would alter characteristics of [3H]dofetilide binding. under physiological conditions (Na+ 135 mM, K+ 5 mM), [3H]dofetilide bound to two sites on guinea-pig ventricular myocytes (a high-affinity site, K(d) 26+/-8 nM, B(max) 81+/-12 fmol/10(6) cells: and a low-affinity site, K(d) 1.6+/-0.8 microM, B(max) 1003+/-173 fmol/10(6) cells, n=11). Binding properties were not altered by changes in osmolarity or extracellular sodium. However, when extracellular K+ was increased to 20 mM, a single binding site was observed with an affinity K(d) of 120+12 nM and a B(max) of 303+/-57 fmol/10(6) cells (P<0.05; n=6). To establish whether this effect was mediated at the high-affinity site we assessed the effects of elevated extracellular potassium on a biological model, neonatal mouse myocytes, that expressed solely the high-affinity binding sites. The K(d) values for binding to fetal mouse cardiac myocytes at an extracellular K+ of 5 mM and 20 mM were also significantly different, 29+/-10 and 230+/-46 nM, respectively. In conclusion, [3H]dofetilide binding to its high-affinity site is modulated by extracellular potassium.
p38 MAPK Isoforms mRNA Protein Heart Hypertrophy Chronic pressure overload p38 mitogen-activated ... more p38 MAPK Isoforms mRNA Protein Heart Hypertrophy Chronic pressure overload p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: α, β, γ, and δ. To date, the prominent isoform in heart has been thought to be p38α. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38γ and p38δ were expressed at protein levels comparable to p38α and 38β, respectively. In the early phase of pressure-overload hypertrophy (1-7 days after constriction of the transverse aorta), the abundance of p38β, p38γ and p38δ mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38α and p38γ in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7-28 days after constriction of the transverse aorta), p38γ immunoreactivity accumulated in the nucleus whereas the distribution of p38α remained unaffected. Hence, both p38α and p38γ are prominent p38 isoforms in heart and p38γ may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.
In most cases, the underlying cause of death is the rapid onset of lethal ventricular arrhythmias... more In most cases, the underlying cause of death is the rapid onset of lethal ventricular arrhythmias in the setting of acquired heart disease. The onset of sudden cardiac death is multifactorial, and the elucidation of the precise molecular and cellular pathways responsible for this phenotype has been difficult due to the paucity of ge-Robert G. Gourdie, 3 Marc M. Rahme, 2 netic insights into this complex human cardiac disease. ). However, studies of affected families have re-La Jolla, California 92093 vealed that only 20% of the patients who harbor the 3 Department of Cell Biology and Anatomy disease genotype display sudden death (Roden and Medical University of South Carolina Spooner, 1999), suggesting the requirement of addi-Charleston, South Carolina 29425 tional pathways that must be present to produce the 4 University of Calgary disease phenotype. Further evidence of the requirement Calgary T2N 4N1 of a "second hit" to produce lethal ventricular arrhyth-Canada mogenesis has come from studies of genetically engineered mice that harbor mutations in the cardiac Na ϩ or K ϩ channels that produce long QT syndrome, prolonged Summary cardiac repolarization, and associated arrhythmias, but not sudden cardiac death (Barry et al., 1998; Charpentier HF-1b, an SP1-related transcription factor, is preferenet al., 1998; Drici et al., 1998; London et al., 1998; Abbott tially expressed in the cardiac conduction system and et al., 1999; Kupershmidt et al., 1999). Currently, there ventricular myocytes in the heart. Mice deficient for
Sex differences in cardiac electrophysiological properties and arrhythmias are evident in epidemi... more Sex differences in cardiac electrophysiological properties and arrhythmias are evident in epidemiologic and investigative studies as well as in daily patient care. At the supraventricular level, women are at increased risk of sick sinus syndrome and atrioventricular (AV) node re-entrant tachycardia, whereas men manifest more AV block and accessory pathway-mediated arrhythmias. At the ventricular level, women are generally at higher risk of long QT-associated arrhythmias, whereas men are more likely to present with early repolarization, idiopathic ventricular fibrillation, and Brugada syndromes. Great advances have been made in unraveling the fundamental mechanisms underlying sex differences in ventricular arrhythmias, particularly those associated with abnormal repolarization. Conversely, the basis for male-predominant arrhythmia risk in structural heart disease and differences in supraventricular arrhythmia susceptibility are poorly understood. Beyond biological differences, arrhythmia occurrence and patient care decisions are also influenced by gender-related factors. This article reviews the current knowledge regarding the nature and underlying mechanisms of sex differences in basic cardiac electrophysiology and clinical arrhythmias.
In this study, we report diVerent protocols used to obtain highly enriched and well-characterized... more In this study, we report diVerent protocols used to obtain highly enriched and well-characterized protein fractions that could be used to determine the subcellular localization of proteins. DiVerent protein fractions (total, cytosolic, total membrane, sarcolemmal, and nuclear) were isolated from mouse heart by a combination of either polytron homogenization or liquid nitrogen pulverization followed by density gradient centrifugation. Triton X-100 was used in speciWc fractions to help in the solubilization of proteins obtained with fractionation protocols. Following the isolation, enzymatic assays and Western blot analysis were used to evaluate the enrichment and/or cross-contamination of these protein fractions. Glucose-6-phosphate dehydrogenase, Na + /K + -ATPase, mitochondrial Ca 2+ -ATPase, sarco-endoplasmic reticulum Ca 2+ -ATPase, glucose-regulated protein, and nucleoporin P62 were used as speciWc markers for the cytosol, sarcolemma, mitochondria, sarco-endoplasmic reticulum, endoplasmic reticulum, and nucleus, respectively. The results show that we obtained enriched protein fractions with little to no crosscontamination. These puriWcation protocols allow us to obtain diVerent protein fractions that could be used in a wide variety of studies.
MK5, a member of the MAPK-activated protein kinase family, is highly expressed in heart. Whereas ... more MK5, a member of the MAPK-activated protein kinase family, is highly expressed in heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart. mRNA for 5 splice variants (MK5.1-5.5), including the original form (MK5.1), was detected. MK5 comprises 14 exons: exon 12 splicing was modified in MK5.2, MK5.3, and MK5.5. MK5.2 and MK5.5 lacked 6 bases at the 3′-end of exon 12, whereas MK5.3 lacked exon 12, resulting in a frame-shift and premature termination of translation at codon 3 of exon 13. MK5.4 and MK5.5 lacked exons 2-6, encoding kinase subdomains IVI, and were kinase-dead. All 5 MK5 variants were detected at the mRNA level in all mouse tissues examined; however, their relative abundance was tissue-specific. Furthermore, the relative abundance of variant mRNA was altered both during hypertrophy and postnatal cardiac development, suggesting the generation or the stability of MK5 variant mRNAs is subject to regulation. When expressed in HEK293 cells, MK5.1, MK5.2 and MK5.3 were nuclear whereas MK5.4 and MK5.5 were cytoplasmic. A p38 MAPK activator, anisomycin, induced the redistribution of each variant. In contrast, MK5 co-immunoprecipitated ERK3, but not ERK4 or p38α, in control and hypertrophying hearts. GST pull-down assays revealed unbound ERK4 and p38α but no free MK5 or ERK3 in heart lysates. Hence, 1) in heart MK5 complexes with ERK3 and 2) MK5 splice variants may mediate distinct effects thus increasing the functional diversity of ERK3-MK5 signaling.
Uploads
Papers by Celine Fiset