The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Robert Horowits - One of the best experts on this subject based on the ideXlab platform.
-
Scaffolds and chaperones in Myofibril assembly: putting the striations in striated muscle
Biophysical Reviews, 2011Co-Authors: Garland L. Crawford, Robert HorowitsAbstract:Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature Myofibrils. These studies led to the view that actin and α-actinin assemble into preMyofibril structures separately from myosin filaments, and that these structures are then assembled into Myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of preMyofibril structures to form mature striated Myofibrils. The evidence shows that Myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of Myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.
-
Myofibril assembly visualized by imaging n rap alpha actinin and actin in living cardiomyocytes
Experimental Cell Research, 2009Co-Authors: Shyam M Manisastry, Kristien J M Zaal, Robert HorowitsAbstract:N-RAP is a striated muscle-specific scaffolding protein that organizes α-actinin and actin into symetrical I-Z-I structures in developing Myofibrils. Here we determined the order of events during Myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either α-actinin or actin. During de novo Myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of α-actinin subsequently organizing along these structures. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The α-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the α-actinin dynamically exchanged during all stages of Myofibril assembly. In contrast, less than 20% of the N-RAP in preMyofibrils was exchanged during 10-20 minutes after photobleaching, but this value increased to 70% during Myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before α-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.
-
krp1 sarcosin promotes lateral fusion of Myofibril assembly intermediates in cultured mouse cardiomyocytes
Experimental Cell Research, 2008Co-Authors: Cynthia C Greenberg, Patricia S Connelly, Mathew P Daniels, Robert HorowitsAbstract:Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of Myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of Myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature Myofibrils, but fibrils remained thin and separated. These thin Myofibrils were degraded by a scission mechanism distinct from the Myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide Myofibrils and contribute insight into mechanisms of Myofibrillogenesis and disassembly.
-
targeted disruption of n rap gene function by rna interference a role for n rap in Myofibril organization
Cytoskeleton, 2006Co-Authors: Ashwini S Dhume, Shajia Lu, Robert HorowitsAbstract:N-RAP is a muscle-specific protein concentrated in Myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by ∼70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching ∼50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased Myofibril assembly, as assessed by α-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature Myofibrils, such as α-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and α-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in Myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that α-actinin organization into Myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms. Cell Motil. Cytoskeleton 2006. Published 2006 Wiley-Liss, Inc.
-
new n rap binding partners alpha actinin filamin and krp1 detected by yeast two hybrid screening implications for Myofibril assembly
Journal of Cell Science, 2003Co-Authors: Shajia Lu, Stefanie Carroll, Amy H Herrera, Bradford W Ozanne, Robert HorowitsAbstract:RAP-IB). We detected significant α-actinin binding to NRAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During Myofibril assembly in cultured chick cardiomyocytes, NRAP and filamin appear to co-localize with α-actinin in the earliest Myofibril precursors found near the cell periphery, as well as in the nascent Myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of Myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during Myofibril assembly. Summary
Steven B Marston - One of the best experts on this subject based on the ideXlab platform.
-
instrumentation to study Myofibril mechanics from static to artificial simulations of cardiac cycle
MethodsX, 2016Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Steven B MarstonAbstract:Abstract Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable Myofibrils can be obtained from frozen tissue. • We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single Myofibrils. The control of Myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently. • This single Myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, Myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure Myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.
-
dcm mutation actce361g causes uncoupling of Myofibril sensitivity from tni phosphorylation that can be reversed by epigallocatechin 3 gallate
Biophysical Journal, 2015Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Weihua Song, Ross Wilkinson, Oneal Copeland, Steven B MarstonAbstract:We examined the relationships between troponin I phosphorylation and Ca2+-regulation of contractility in single Myofibrils from a mouse model of familial DCM (ACTC E361G) in comparison with non-transgenic (NTG). We measured the effects of changing [Ca2+] and troponin I phosphorylation level on Myofibril contractility. Propranolol treatment of mice was used to reduce the level of troponin I in their hearts prior to isolating the Myofibrils.For non-transgenic mouse Myofibrils we found that when the TnI phosphorylation level was reduced from 1.02 to 0.3 the Ca2+-sensitivity of force was increased (EC50P/unP=1.8), relaxation parameter kREL was reduced and tLIN was increased. ACTCE361G mouse Myofibrils were uncoupled: Ca2+-sensitivity and relaxation parameters did not depend on troponin I phosphorylation level (EC50P/unP=0.88). Nevertheless, modulation of Ca2+-sensitivity by sarcomere length or due to EMD57033 was retained.The Ca2+-desensitiser Epigallocatechin-3-gallate (EGCG) decreased Ca2+-sensitivity in phosphorylated and unphosphorylated NTG Myofibrils equally (EC50 P/uP= 0.50±0.06 and 0.45±0.07 respectively) but did not change the relaxation parameters tLIN and kRE. The rate of force development (kACT), measured at high Ca2+, was unchanged in Myofibrils with phosphorylated TnI and 22% decreased in unphosphorylated Myofibrils indicating that EGCG affects cross-bridge activation kinetics.EGCG reduced Ca2+-sensitivity and kACT in both phosphorylated and unphosphorylated ACTCE361G Myofibrils. The change in EC50 was more in phosphorylated than unphosphorylated Myofibrils, consequently EGCG restored the lost difference in EC50 values between phosphorylated and unphosphorylated Myofibrils and also the difference in relaxation parameters tLIN and kREL. The observation that EGCG does not affect either the EC50 P/ EC50 unP or tLIN in NTG Myofibrils but changes them in ACTG E361G suggests that EGCG can restore modulation of cardiac contractile function by TnI phosphorylation to DCM mutant Myofibrils independently of its Ca2+-desensitising function.
-
dcm causing mutation e361g in actin uncouples Myofibril ca2 sensitivity from protein phosphorylation
Biophysical Journal, 2014Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Weihua Song, Ross Wilkinson, Oneal Copeland, Steven B MarstonAbstract:Experiments using the in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation of troponin I and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle Myofibrils producing isometric force. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.1 mol Pi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of propranolol which reduced of both TnI bisphosphorylation and MyBP-C phosphorylation by at least 95%. Then we measured contraction in single Myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The Ca2+-sensitivity of isometric force and the kinetics of tension development and relaxation for the ACTC E361G TG mouse Myofibrils were compared with WT. Modulation of the Ca2+-sensitivity by changes in sarcomere length and by the Ca2+-sensitizer EMD 57033 was further investigated.The maximum isometric force and the tension development rate was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse Myofibrils. The ACTC E361G Myofibrils had higher Ca2+-sensitivity of force development then WT. Despite that, the magnitude of length dependent activation was well preserved in ACTC E361G Myofibrils. Essentially, while the Ca2+-sensitivity of isometric force was increased 1.4-fold when WT Myofibrils were dephosphorylated, the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. Additionally, comparing dephosphorylated WT vs WT, the relaxation rate was 1.3-fold slower but dephosphorylation did not change the kinetics of E361G Myofibril. Changes in Ca2+-sensitivity were correlated with changes in relaxation rate. Thus the ACTC E361G mutation uncoupled Ca2+-sensitivity and lusitropy from the level of TnI and MyBP-C phosphorylation in intact Myofibrils.
-
dcm causing mutation e361g in actin slows Myofibril relaxation kinetics and uncouples Myofibril ca2 sensitivity from protein phosphorylation
Biophysical Journal, 2013Co-Authors: Petr G Vikhorev, Steven B Marston, Weihua Song, Ross Wilkinson, Neal O Copeland, Michael A FerencziAbstract:Previous experiments using the unloaded in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation by PKA of troponin I (Ser 22 and 23) and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle Myofibrils producing isometric force. Myofibrils were isolated from wild-type and ACTC E361G mouse hearts. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.4 molPi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of beta-adrenoceptor agonist propranolol which reduced phosphorylation of both TnI and MyBP-C by 70-80-%. Myofibrils were isolated from propranolol-treated and untreated mouse hearts. Then we measured contraction in single Myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The maximum isometric force was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse Myofibrils. The duration of the linear slow phase of relaxation was longer (1.4-1.8 fold) and the rate of the fast phase was reduced (1.1-1.4 fold) for the ACTC E361G TG compared to WT. The Ca2+-sensitivity of isometric force was increased 1.4-fold (p=0.021) when WT Myofibrils were dephosphorylated. In contrast the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. This pattern was also apparent in the rates of relaxation. Comparing dephosphorylated WT vs WT, the duration of the linear slow phase was longer and the rate of the fast phase was reduced but dephosphorylation did not change the kinetics of E361G Myofibril. Thus the ACTC E361G mutation fully uncoupled Ca2+ sensitivity from the level of TnI and MyBP-C phosphorylation in intact Myofibrils as we have seen with IVMA.
Petr G Vikhorev - One of the best experts on this subject based on the ideXlab platform.
-
instrumentation to study Myofibril mechanics from static to artificial simulations of cardiac cycle
MethodsX, 2016Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Steven B MarstonAbstract:Abstract Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable Myofibrils can be obtained from frozen tissue. • We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single Myofibrils. The control of Myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently. • This single Myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, Myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure Myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.
-
dcm mutation actce361g causes uncoupling of Myofibril sensitivity from tni phosphorylation that can be reversed by epigallocatechin 3 gallate
Biophysical Journal, 2015Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Weihua Song, Ross Wilkinson, Oneal Copeland, Steven B MarstonAbstract:We examined the relationships between troponin I phosphorylation and Ca2+-regulation of contractility in single Myofibrils from a mouse model of familial DCM (ACTC E361G) in comparison with non-transgenic (NTG). We measured the effects of changing [Ca2+] and troponin I phosphorylation level on Myofibril contractility. Propranolol treatment of mice was used to reduce the level of troponin I in their hearts prior to isolating the Myofibrils.For non-transgenic mouse Myofibrils we found that when the TnI phosphorylation level was reduced from 1.02 to 0.3 the Ca2+-sensitivity of force was increased (EC50P/unP=1.8), relaxation parameter kREL was reduced and tLIN was increased. ACTCE361G mouse Myofibrils were uncoupled: Ca2+-sensitivity and relaxation parameters did not depend on troponin I phosphorylation level (EC50P/unP=0.88). Nevertheless, modulation of Ca2+-sensitivity by sarcomere length or due to EMD57033 was retained.The Ca2+-desensitiser Epigallocatechin-3-gallate (EGCG) decreased Ca2+-sensitivity in phosphorylated and unphosphorylated NTG Myofibrils equally (EC50 P/uP= 0.50±0.06 and 0.45±0.07 respectively) but did not change the relaxation parameters tLIN and kRE. The rate of force development (kACT), measured at high Ca2+, was unchanged in Myofibrils with phosphorylated TnI and 22% decreased in unphosphorylated Myofibrils indicating that EGCG affects cross-bridge activation kinetics.EGCG reduced Ca2+-sensitivity and kACT in both phosphorylated and unphosphorylated ACTCE361G Myofibrils. The change in EC50 was more in phosphorylated than unphosphorylated Myofibrils, consequently EGCG restored the lost difference in EC50 values between phosphorylated and unphosphorylated Myofibrils and also the difference in relaxation parameters tLIN and kREL. The observation that EGCG does not affect either the EC50 P/ EC50 unP or tLIN in NTG Myofibrils but changes them in ACTG E361G suggests that EGCG can restore modulation of cardiac contractile function by TnI phosphorylation to DCM mutant Myofibrils independently of its Ca2+-desensitising function.
-
dcm causing mutation e361g in actin uncouples Myofibril ca2 sensitivity from protein phosphorylation
Biophysical Journal, 2014Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Weihua Song, Ross Wilkinson, Oneal Copeland, Steven B MarstonAbstract:Experiments using the in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation of troponin I and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle Myofibrils producing isometric force. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.1 mol Pi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of propranolol which reduced of both TnI bisphosphorylation and MyBP-C phosphorylation by at least 95%. Then we measured contraction in single Myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The Ca2+-sensitivity of isometric force and the kinetics of tension development and relaxation for the ACTC E361G TG mouse Myofibrils were compared with WT. Modulation of the Ca2+-sensitivity by changes in sarcomere length and by the Ca2+-sensitizer EMD 57033 was further investigated.The maximum isometric force and the tension development rate was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse Myofibrils. The ACTC E361G Myofibrils had higher Ca2+-sensitivity of force development then WT. Despite that, the magnitude of length dependent activation was well preserved in ACTC E361G Myofibrils. Essentially, while the Ca2+-sensitivity of isometric force was increased 1.4-fold when WT Myofibrils were dephosphorylated, the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. Additionally, comparing dephosphorylated WT vs WT, the relaxation rate was 1.3-fold slower but dephosphorylation did not change the kinetics of E361G Myofibril. Changes in Ca2+-sensitivity were correlated with changes in relaxation rate. Thus the ACTC E361G mutation uncoupled Ca2+-sensitivity and lusitropy from the level of TnI and MyBP-C phosphorylation in intact Myofibrils.
-
dcm causing mutation e361g in actin slows Myofibril relaxation kinetics and uncouples Myofibril ca2 sensitivity from protein phosphorylation
Biophysical Journal, 2013Co-Authors: Petr G Vikhorev, Steven B Marston, Weihua Song, Ross Wilkinson, Neal O Copeland, Michael A FerencziAbstract:Previous experiments using the unloaded in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation by PKA of troponin I (Ser 22 and 23) and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle Myofibrils producing isometric force. Myofibrils were isolated from wild-type and ACTC E361G mouse hearts. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.4 molPi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of beta-adrenoceptor agonist propranolol which reduced phosphorylation of both TnI and MyBP-C by 70-80-%. Myofibrils were isolated from propranolol-treated and untreated mouse hearts. Then we measured contraction in single Myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The maximum isometric force was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse Myofibrils. The duration of the linear slow phase of relaxation was longer (1.4-1.8 fold) and the rate of the fast phase was reduced (1.1-1.4 fold) for the ACTC E361G TG compared to WT. The Ca2+-sensitivity of isometric force was increased 1.4-fold (p=0.021) when WT Myofibrils were dephosphorylated. In contrast the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. This pattern was also apparent in the rates of relaxation. Comparing dephosphorylated WT vs WT, the duration of the linear slow phase was longer and the rate of the fast phase was reduced but dephosphorylation did not change the kinetics of E361G Myofibril. Thus the ACTC E361G mutation fully uncoupled Ca2+ sensitivity from the level of TnI and MyBP-C phosphorylation in intact Myofibrils as we have seen with IVMA.
Michael A Ferenczi - One of the best experts on this subject based on the ideXlab platform.
-
instrumentation to study Myofibril mechanics from static to artificial simulations of cardiac cycle
MethodsX, 2016Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Steven B MarstonAbstract:Abstract Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable Myofibrils can be obtained from frozen tissue. • We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single Myofibrils. The control of Myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently. • This single Myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, Myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure Myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.
-
dcm mutation actce361g causes uncoupling of Myofibril sensitivity from tni phosphorylation that can be reversed by epigallocatechin 3 gallate
Biophysical Journal, 2015Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Weihua Song, Ross Wilkinson, Oneal Copeland, Steven B MarstonAbstract:We examined the relationships between troponin I phosphorylation and Ca2+-regulation of contractility in single Myofibrils from a mouse model of familial DCM (ACTC E361G) in comparison with non-transgenic (NTG). We measured the effects of changing [Ca2+] and troponin I phosphorylation level on Myofibril contractility. Propranolol treatment of mice was used to reduce the level of troponin I in their hearts prior to isolating the Myofibrils.For non-transgenic mouse Myofibrils we found that when the TnI phosphorylation level was reduced from 1.02 to 0.3 the Ca2+-sensitivity of force was increased (EC50P/unP=1.8), relaxation parameter kREL was reduced and tLIN was increased. ACTCE361G mouse Myofibrils were uncoupled: Ca2+-sensitivity and relaxation parameters did not depend on troponin I phosphorylation level (EC50P/unP=0.88). Nevertheless, modulation of Ca2+-sensitivity by sarcomere length or due to EMD57033 was retained.The Ca2+-desensitiser Epigallocatechin-3-gallate (EGCG) decreased Ca2+-sensitivity in phosphorylated and unphosphorylated NTG Myofibrils equally (EC50 P/uP= 0.50±0.06 and 0.45±0.07 respectively) but did not change the relaxation parameters tLIN and kRE. The rate of force development (kACT), measured at high Ca2+, was unchanged in Myofibrils with phosphorylated TnI and 22% decreased in unphosphorylated Myofibrils indicating that EGCG affects cross-bridge activation kinetics.EGCG reduced Ca2+-sensitivity and kACT in both phosphorylated and unphosphorylated ACTCE361G Myofibrils. The change in EC50 was more in phosphorylated than unphosphorylated Myofibrils, consequently EGCG restored the lost difference in EC50 values between phosphorylated and unphosphorylated Myofibrils and also the difference in relaxation parameters tLIN and kREL. The observation that EGCG does not affect either the EC50 P/ EC50 unP or tLIN in NTG Myofibrils but changes them in ACTG E361G suggests that EGCG can restore modulation of cardiac contractile function by TnI phosphorylation to DCM mutant Myofibrils independently of its Ca2+-desensitising function.
-
dcm causing mutation e361g in actin uncouples Myofibril ca2 sensitivity from protein phosphorylation
Biophysical Journal, 2014Co-Authors: Petr G Vikhorev, Michael A Ferenczi, Weihua Song, Ross Wilkinson, Oneal Copeland, Steven B MarstonAbstract:Experiments using the in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation of troponin I and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle Myofibrils producing isometric force. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.1 mol Pi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of propranolol which reduced of both TnI bisphosphorylation and MyBP-C phosphorylation by at least 95%. Then we measured contraction in single Myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The Ca2+-sensitivity of isometric force and the kinetics of tension development and relaxation for the ACTC E361G TG mouse Myofibrils were compared with WT. Modulation of the Ca2+-sensitivity by changes in sarcomere length and by the Ca2+-sensitizer EMD 57033 was further investigated.The maximum isometric force and the tension development rate was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse Myofibrils. The ACTC E361G Myofibrils had higher Ca2+-sensitivity of force development then WT. Despite that, the magnitude of length dependent activation was well preserved in ACTC E361G Myofibrils. Essentially, while the Ca2+-sensitivity of isometric force was increased 1.4-fold when WT Myofibrils were dephosphorylated, the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. Additionally, comparing dephosphorylated WT vs WT, the relaxation rate was 1.3-fold slower but dephosphorylation did not change the kinetics of E361G Myofibril. Changes in Ca2+-sensitivity were correlated with changes in relaxation rate. Thus the ACTC E361G mutation uncoupled Ca2+-sensitivity and lusitropy from the level of TnI and MyBP-C phosphorylation in intact Myofibrils.
-
dcm causing mutation e361g in actin slows Myofibril relaxation kinetics and uncouples Myofibril ca2 sensitivity from protein phosphorylation
Biophysical Journal, 2013Co-Authors: Petr G Vikhorev, Steven B Marston, Weihua Song, Ross Wilkinson, Neal O Copeland, Michael A FerencziAbstract:Previous experiments using the unloaded in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation by PKA of troponin I (Ser 22 and 23) and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle Myofibrils producing isometric force. Myofibrils were isolated from wild-type and ACTC E361G mouse hearts. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.4 molPi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of beta-adrenoceptor agonist propranolol which reduced phosphorylation of both TnI and MyBP-C by 70-80-%. Myofibrils were isolated from propranolol-treated and untreated mouse hearts. Then we measured contraction in single Myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The maximum isometric force was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse Myofibrils. The duration of the linear slow phase of relaxation was longer (1.4-1.8 fold) and the rate of the fast phase was reduced (1.1-1.4 fold) for the ACTC E361G TG compared to WT. The Ca2+-sensitivity of isometric force was increased 1.4-fold (p=0.021) when WT Myofibrils were dephosphorylated. In contrast the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. This pattern was also apparent in the rates of relaxation. Comparing dephosphorylated WT vs WT, the duration of the linear slow phase was longer and the rate of the fast phase was reduced but dephosphorylation did not change the kinetics of E361G Myofibril. Thus the ACTC E361G mutation fully uncoupled Ca2+ sensitivity from the level of TnI and MyBP-C phosphorylation in intact Myofibrils as we have seen with IVMA.
Velia M Fowler - One of the best experts on this subject based on the ideXlab platform.
-
aberrant Myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality
Journal of Cell Biology, 2003Co-Authors: Kimberly L Fritzsix, Robert S Fischer, Bisong Xu, Carol C Gregorio, Huda Y Zoghbi, Velia M FowlerAbstract:Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated Myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8–8.5 Tmod1 null embryos revealed structures resembling nascent Myofibrils with continuous F-actin staining and periodic dots of α-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, Myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and Myofibril maturation; this is critical for heart morphogenesis during embryonic development.
-
thin filaments elongate from their pointed ends during Myofibril assembly in drosophila indirect flight muscle
Journal of Cell Biology, 2001Co-Authors: Michelle Mardahldumesnil, Velia M FowlerAbstract:Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled Myofibrils, its role in length specification during de novo Myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo ( spdo ), to investigate Tmod9s function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout Myofibril assembly. Transient overexpression of SPDO during Myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during Myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during Myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during Myofibril assembly is crucial for specification of final thin filament lengths, Myofibril structure, and muscle function.
