The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform
Thomas P. Burghardt - One of the best experts on this subject based on the ideXlab platform.
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Analytical comparison of natural and pharmaceutical Ventricular Myosin activators.
Biochemistry, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Ventricular Myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, Myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive Myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated Myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive Myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural Myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.
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Ventricular Myosin modifies in vitro step size when phosphorylated
Journal of Molecular and Cellular Cardiology, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Cardiac and skeletal muscle Myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The Myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to Myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5 nm step-size for fast skeletal Myosin and multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement for β cardiac Myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8 nm step frequency. After phosphorylation, the 8 nm step is the dominant Myosin step-size resulting in significant gain in the average step-size. An increase in Myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the Myosin filament.
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Ventricular Myosin Modifies in Vitro Step-Size When Phosphorylated
Biophysical Journal, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Cardiac and skeletal muscle Myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin in transduction/mechanical coupling. Inheritable cardiomyopathies are more frequently linked to Myosin mutations than other sarcomeric proteins. Hereditary skeletal myopathies linked to Myosin are less common. They lead to muscle weakness or affect Myosin isoforms expressed during development leading to arthrogryposis syndromes. Myosin has a motor domain containing ATP and actin binding sites and light chains stabilized lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into linear displacement (step-size). Relative Myosin and actin filament sliding is modeled in vitro with a motility assay quantitating actin filament translation over a Myosin coated surface. A novel quantum dot super-resolution in vitro motility assay confirmed a 5 nm step-size for fast skeletal Myosin while β cardiac Myosin (βMys) had multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement. The Myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to disease and to Myosin filament based contraction regulation. We have studied the effect of RLC phosphorylation on the step-size of porcine βMys. Phosphorylated βMys has ∼85% of the Myosin phosphorylated. We find RLC phosphorylation causing the distribution of longest step increasing from 37% to 71%. This dramatic re-distribution of step-sizes provides significant gain in average step-size. The results indicate a mechanism for contraction regulation by step-size adaptation using post-translational modification of the Myosin filament via RLC phosphorylation. Research supported by R01AR049277, R01HL095572 and the Mayo Foundation.
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Light Chain Kinase Specificity in Cardiac Myosin
Biophysical Journal, 2012Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Human Ventricular cardiac Myosin regulatory light chain (MYL2) phosphorylation modifies Ser15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle Myosin light chain kinase (smMLCK) is prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated Ser15 in MYL2 in vitro. Specific modification of Ser15 was verified by direct detection of the phospho group on Ser15 with mass spectrometry. SmMLCK also specifically phosphorylated Myosin regulatory light chain Ser15 in porcine Ventricular Myosin and chicken gizzard smooth muscle Myosin (Ser20 in smooth muscle) but failed to phosphorylate the Myosin regulatory light chain in rabbit skeletal Myosin. Michaelis-Menten Vm and KM constants for Ser15 phosphorylation in MYL2, porcine Ventricular Myosin, and chicken gizzard Myosin are similar. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle Myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.
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Smooth muscle Myosin light chain kinase efficiently phosphorylates serine 15 of cardiac Myosin regulatory light chain.
Biochemical and biophysical research communications, 2011Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Specific phosphorylation of the human Ventricular cardiac Myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle Myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated Myosin regulatory light chain S15 in porcine Ventricular Myosin and chicken gizzard smooth muscle Myosin (S20 in smooth muscle) but failed to phosphorylate the Myosin regulatory light chain in rabbit skeletal Myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine Ventricular Myosin, and chicken gizzard Myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle Myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
Katalin Ajtai - One of the best experts on this subject based on the ideXlab platform.
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Analytical comparison of natural and pharmaceutical Ventricular Myosin activators.
Biochemistry, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Ventricular Myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, Myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive Myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated Myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive Myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural Myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.
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Ventricular Myosin modifies in vitro step size when phosphorylated
Journal of Molecular and Cellular Cardiology, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Cardiac and skeletal muscle Myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The Myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to Myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5 nm step-size for fast skeletal Myosin and multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement for β cardiac Myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8 nm step frequency. After phosphorylation, the 8 nm step is the dominant Myosin step-size resulting in significant gain in the average step-size. An increase in Myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the Myosin filament.
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Ventricular Myosin Modifies in Vitro Step-Size When Phosphorylated
Biophysical Journal, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Cardiac and skeletal muscle Myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin in transduction/mechanical coupling. Inheritable cardiomyopathies are more frequently linked to Myosin mutations than other sarcomeric proteins. Hereditary skeletal myopathies linked to Myosin are less common. They lead to muscle weakness or affect Myosin isoforms expressed during development leading to arthrogryposis syndromes. Myosin has a motor domain containing ATP and actin binding sites and light chains stabilized lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into linear displacement (step-size). Relative Myosin and actin filament sliding is modeled in vitro with a motility assay quantitating actin filament translation over a Myosin coated surface. A novel quantum dot super-resolution in vitro motility assay confirmed a 5 nm step-size for fast skeletal Myosin while β cardiac Myosin (βMys) had multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement. The Myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to disease and to Myosin filament based contraction regulation. We have studied the effect of RLC phosphorylation on the step-size of porcine βMys. Phosphorylated βMys has ∼85% of the Myosin phosphorylated. We find RLC phosphorylation causing the distribution of longest step increasing from 37% to 71%. This dramatic re-distribution of step-sizes provides significant gain in average step-size. The results indicate a mechanism for contraction regulation by step-size adaptation using post-translational modification of the Myosin filament via RLC phosphorylation. Research supported by R01AR049277, R01HL095572 and the Mayo Foundation.
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Light Chain Kinase Specificity in Cardiac Myosin
Biophysical Journal, 2012Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Human Ventricular cardiac Myosin regulatory light chain (MYL2) phosphorylation modifies Ser15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle Myosin light chain kinase (smMLCK) is prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated Ser15 in MYL2 in vitro. Specific modification of Ser15 was verified by direct detection of the phospho group on Ser15 with mass spectrometry. SmMLCK also specifically phosphorylated Myosin regulatory light chain Ser15 in porcine Ventricular Myosin and chicken gizzard smooth muscle Myosin (Ser20 in smooth muscle) but failed to phosphorylate the Myosin regulatory light chain in rabbit skeletal Myosin. Michaelis-Menten Vm and KM constants for Ser15 phosphorylation in MYL2, porcine Ventricular Myosin, and chicken gizzard Myosin are similar. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle Myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.
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Smooth muscle Myosin light chain kinase efficiently phosphorylates serine 15 of cardiac Myosin regulatory light chain.
Biochemical and biophysical research communications, 2011Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Specific phosphorylation of the human Ventricular cardiac Myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle Myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated Myosin regulatory light chain S15 in porcine Ventricular Myosin and chicken gizzard smooth muscle Myosin (S20 in smooth muscle) but failed to phosphorylate the Myosin regulatory light chain in rabbit skeletal Myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine Ventricular Myosin, and chicken gizzard Myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle Myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
Yihua Wang - One of the best experts on this subject based on the ideXlab platform.
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Analytical comparison of natural and pharmaceutical Ventricular Myosin activators.
Biochemistry, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Ventricular Myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, Myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive Myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated Myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive Myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural Myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.
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Ventricular Myosin modifies in vitro step size when phosphorylated
Journal of Molecular and Cellular Cardiology, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Cardiac and skeletal muscle Myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The Myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to Myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5 nm step-size for fast skeletal Myosin and multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement for β cardiac Myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8 nm step frequency. After phosphorylation, the 8 nm step is the dominant Myosin step-size resulting in significant gain in the average step-size. An increase in Myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the Myosin filament.
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Ventricular Myosin Modifies in Vitro Step-Size When Phosphorylated
Biophysical Journal, 2014Co-Authors: Yihua Wang, Katalin Ajtai, Thomas P. BurghardtAbstract:Cardiac and skeletal muscle Myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin in transduction/mechanical coupling. Inheritable cardiomyopathies are more frequently linked to Myosin mutations than other sarcomeric proteins. Hereditary skeletal myopathies linked to Myosin are less common. They lead to muscle weakness or affect Myosin isoforms expressed during development leading to arthrogryposis syndromes. Myosin has a motor domain containing ATP and actin binding sites and light chains stabilized lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into linear displacement (step-size). Relative Myosin and actin filament sliding is modeled in vitro with a motility assay quantitating actin filament translation over a Myosin coated surface. A novel quantum dot super-resolution in vitro motility assay confirmed a 5 nm step-size for fast skeletal Myosin while β cardiac Myosin (βMys) had multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement. The Myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to disease and to Myosin filament based contraction regulation. We have studied the effect of RLC phosphorylation on the step-size of porcine βMys. Phosphorylated βMys has ∼85% of the Myosin phosphorylated. We find RLC phosphorylation causing the distribution of longest step increasing from 37% to 71%. This dramatic re-distribution of step-sizes provides significant gain in average step-size. The results indicate a mechanism for contraction regulation by step-size adaptation using post-translational modification of the Myosin filament via RLC phosphorylation. Research supported by R01AR049277, R01HL095572 and the Mayo Foundation.
Hans R. Figulla - One of the best experts on this subject based on the ideXlab platform.
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Identification and validation of selective upregulation of Ventricular Myosin light chain type 2 mRNA in idiopathic dilated cardiomyopathy.
European journal of heart failure, 2002Co-Authors: Daniela Haase, Michael H. Lehmann, M. M. Körner, Reiner Körfer, Holger H. Sigusch, Hans R. FigullaAbstract:Background and aims: the etiology of idiopathic dilated cardiomyopathy (IDCM) is unknown, methods such as suppression subtractive hybridization (SSH) and DNA microarray technology can help to identify genes which might be involved in the pathogenesis of this disease. Methods and results: we used SSH which compared mRNA populations extracted from the left Ventricular tissue of IDCM hearts and from the control tissue to identify sequences which correspond to genes up-regulated in IDCM. We identified Ventricular Myosin light chain type 2 (MLC2V), skeletal α-actin, long-chain-acyl-CoA-synthetase and mRNA for the protein KIAA0465 as differentially up-regulated genes. Expression of MLC2V mRNA was determined by RT-PCR in patients with end-stage heart failure caused by IDCM (n-11) or coronary artery disease (CAD, n-9) who underwent heart transplantation as well as the controls (n-6). MLC2V/GAPDH ratios were 2.95±0.32, 0.69±0.03 and 0.28±0.08 (arbitrary unit) for the IDCM group, the CAD group and controls, respectively (P
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identification and validation of selective upregulation of Ventricular Myosin light chain type 2 mrna in idiopathic dilated cardiomyopathy
European Journal of Heart Failure, 2002Co-Authors: Daniela Haase, Michael H. Lehmann, M. M. Körner, Reiner Körfer, Holger H. Sigusch, Hans R. FigullaAbstract:Background and aims: the etiology of idiopathic dilated cardiomyopathy (IDCM) is unknown, methods such as suppression subtractive hybridization (SSH) and DNA microarray technology can help to identify genes which might be involved in the pathogenesis of this disease. Methods and results: we used SSH which compared mRNA populations extracted from the left Ventricular tissue of IDCM hearts and from the control tissue to identify sequences which correspond to genes up-regulated in IDCM. We identified Ventricular Myosin light chain type 2 (MLC2V), skeletal α-actin, long-chain-acyl-CoA-synthetase and mRNA for the protein KIAA0465 as differentially up-regulated genes. Expression of MLC2V mRNA was determined by RT-PCR in patients with end-stage heart failure caused by IDCM (n-11) or coronary artery disease (CAD, n-9) who underwent heart transplantation as well as the controls (n-6). MLC2V/GAPDH ratios were 2.95±0.32, 0.69±0.03 and 0.28±0.08 (arbitrary unit) for the IDCM group, the CAD group and controls, respectively (P<0.05). DNA microarray analysis confirmed the finding of MLC2V upregulation in IDCM (3.7- and 1.8-fold increase in MLC2V mRNA). Conclusions: we have demonstrated that SSH is a useful method to identify differential myocardial upregulation of genes. Upregulation of MLC2V can be judged as a specific IDCM related feature, which might be clinically helpful.
Qinwei Shi - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the upstream regulatory region of human Ventricular Myosin light chain 1 gene.
Journal of molecular and cellular cardiology, 1992Co-Authors: Qinwei Shi, Donald A.g. Mickle, G. JackowskiAbstract:Abstract To explore the mechanisms regulating expression of Ventricular Myosin light chain 1, the human gene including 5′-flanking DNA was cloned and characterized by Southern blot and restriction mapping. A 2 kb 5′-flanking DNA was sequenced and linked to a chloramphenicol acetyltransferase reporter gene. The constructs then were transfected into cultured human and rat cardiomyocytes as well as rat aortic endothelial cells. Deletion analysis of constructs revealed that the basal promoter sequences, which were located within 62 base pairs of the cap site, could direct high levels of chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. The region between −62 to −312 base pairs strongly repressed the chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. Positive elements were found between −312 and −2000 base pairs of the cap site. These resuts are indicative, among other possibilities, that the human Ventricular Myosin light chain 1 gene is turned on in cardiomyocytes by the presence of trans-acting factors that are bound to upstream positive elements and is turned off in non-muscle cells by the presence of repressor-binding proteins. But this mechanism remains to be established.
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Expression of Ventricular Myosin subunits in the atria of children with congenital heart malformations.
Circulation research, 1991Co-Authors: Qinwei Shi, U. Danilczyk, Jinxia Wang, Yew Phew See, William G. Williams, G. A. Trusler, R. Beaulieu, V. Rose, George JackowskiAbstract:The presence of Ventricular Myosin light chains in the atria of children with congenital heart disease was demonstrated by two-dimensional polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis. Ventricular Myosin light chains were present in 27% of biopsies from 91 children with different forms of congenital heart disease. Perimembranous Ventricular septal defects and tetralogy of Fallot were associated with the presence of Ventricular Myosin light chains in 50% of patients. The presence of Ventricular Myosin light chains in these atria did not correlate with pressure or volume overload. Analysis of Myosin heavy chain isotype in the same biopsies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis indicated that there was no detectable expression of Ventricular Myosin heavy chain (beta-subunit), suggesting that the genes for the Myosin heavy chains and light chains are not expressed coordinately.
