E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics

2006 ◽  
Vol 291 (5) ◽  
pp. H2098-H2106 ◽  
Author(s):  
D. Dumka ◽  
J. Talent ◽  
I. Akopova ◽  
G. Guzman ◽  
D. Szczesna-Cordary ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Light Chain ◽  
Left Ventricular ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Time Resolved ◽  
Essential Light Chain ◽  
Cross Bridge ◽  
The Difference ◽  
Cross Bridges ◽  
The Times

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca2+-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (τ1) of myosin heads from thin filaments, 2) rebinding time (τ2) of the cross bridges to actin, and 3) dissociation time (τ3) of ADP from the active site of myosin. τ1 was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. τ2 was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. τ3 was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. τ1 was statistically greater in Tg-m than in controls. τ2 was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. τ3 was the same in Tg-m and controls. To determine whether the difference in τ1 was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in τ1 was probably caused by myofibrillar disarray. The decrease in τ2 of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

scholarly journals Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy

2013 ◽  
Vol 305 (4) ◽  
pp. H575-H589 ◽  
Author(s):  
Katarzyna Kazmierczak ◽  
Ellena C. Paulino ◽  
Wenrui Huang ◽  
Priya Muthu ◽  
Jingsheng Liang ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Light Chain ◽  
Left Ventricular ◽  
Compensatory Hypertrophy ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Heart Weight ◽  
Myocardial Stiffness ◽  
Essential Light Chain

The functional consequences of the familial hypertrophic cardiomyopathy A57G (alanine-to-glycine) mutation in the myosin ventricular essential light chain (ELC) were assessed in vitro and in vivo using previously generated transgenic (Tg) mice expressing A57G-ELC mutant vs. wild-type (WT) of human cardiac ELC and in recombinant A57G- or WT-protein-exchanged porcine cardiac muscle strips. Compared with the Tg-WT, there was a significant increase in the Ca2+ sensitivity of force (ΔpCa50 ≅ 0.1) and an ∼1.3-fold decrease in maximal force per cross section of muscle observed in the mutant preparations. In addition, a significant increase in passive tension in response to stretch was monitored in Tg-A57G vs. Tg-WT strips indicating a mutation-induced myocardial stiffness. Consistently, the hearts of Tg-A57G mice demonstrated a high level of fibrosis and hypertrophy manifested by increased heart weight-to-body weight ratios and a decreased number of nuclei indicating an increase in the two-dimensional size of Tg-A57G vs. Tg-WT myocytes. Echocardiography examination showed a phenotype of eccentric hypertrophy in Tg-A57G mice, enhanced left ventricular (LV) cavity dimension without changes in LV posterior/anterior wall thickness. Invasive hemodynamics data revealed significantly increased end-systolic elastance, defined by the slope of the pressure-volume relationship, indicating a mutation-induced increase in cardiac contractility. Our results suggest that the A57G allele causes disease by means of a discrete modulation of myofilament function, increased Ca2+ sensitivity, and decreased maximal tension followed by compensatory hypertrophy and enhanced contractility. These and other contributing factors such as increased myocardial stiffness and fibrosis most likely activate cardiomyopathic signaling pathways leading to pathologic cardiac remodeling.

scholarly journals A Novel Myosin Essential Light Chain Mutation Causes Hypertrophic Cardiomyopathy with Late Onset and Low Expressivity

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Paal Skytt Andersen ◽  
Paula Louise Hedley ◽  
Stephen P. Page ◽  
Petros Syrris ◽  
Johanna Catharina Moolman-Smook ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Light Chain ◽  
Late Onset ◽  
Large Family ◽  
Left Ventricular ◽  
Cascade Screening ◽  
Essential Light Chain ◽  
Mutation Carriers ◽  
Genes Encoding ◽  
The Mean

Hypertrophic cardiomyopathy (HCM) is caused by mutations in genes encoding sarcomere proteins. Mutations inMYL3, encoding the essential light chain of myosin, are rare and have been associated with sudden death. Both recessive and dominant patterns of inheritance have been suggested. We studied a large family with a 38-year-old asymptomatic HCM-affected male referred because of a murmur. The patient had HCM with left ventricular hypertrophy (max WT 21 mm), a resting left ventricular outflow gradient of 36 mm Hg, and left atrial dilation (54 mm). Genotyping revealed heterozygosity for a novel missense mutation, p.V79I, inMYL3. The mutation was not found in 300 controls, and the patient had no mutations in 10 sarcomere genes. Cascade screening revealed a further nine heterozygote mutation carriers, three of whom had ECG and/or echocardiographic abnormalities but did not fulfil diagnostic criteria for HCM. The penetrance, if we consider this borderline HCM the phenotype of the p.V79I mutation, was 40%, but the mean age of the nonpenetrant mutation carriers is 15, while the mean age of the penetrant mutation carriers is 47. The mutation affects a conserved valine replacing it with a larger isoleucine residue in the region of contact between the light chain and the myosin lever arm. In conclusion,MYL3mutations can present with low expressivity and late onset.

scholarly journals Temporal and mutation-specific alterations in Ca2+ homeostasis differentially determine the progression of cTnT-related cardiomyopathies in murine models

2009 ◽  
Vol 297 (2) ◽  
pp. H614-H626 ◽  
Author(s):  
Pia J. Guinto ◽  
Todd E. Haim ◽  
Candice C. Dowell-Martino ◽  
Nathaniel Sibinga ◽  
Jil C. Tardiff
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Ventricular Myocytes ◽  
Troponin T ◽  
Early Time ◽  
Left Ventricular ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Compensatory Mechanism ◽  
Missense Mutations ◽  
Tail Domain ◽  
Cellular Mechanics

Naturally occurring mutations in cardiac troponin T (cTnT) result in a clinical subset of familial hypertrophic cardiomyopathy. To determine the mechanistic links between thin-filament mutations and cardiovascular phenotypes, we have generated and characterized several transgenic mouse models carrying cTnT mutations. We address two central questions regarding the previously observed changes in myocellular mechanics and Ca2+ homeostasis: 1) are they characteristic of all severe cTnT mutations, and 2) are they primary (early) or secondary (late) components of the myocellular response? Adult left ventricular myocytes were isolated from 2- and 6-mo-old transgenic mice carrying missense mutations at residue 92, flanking the TNT1 NH2-terminal tail domain. Results from R92L and R92W myocytes showed mutation-specific alterations in contraction and relaxation indexes at 2 mo with improvements by 6 mo. Alterations in Ca2+ kinetics remained consistent with mechanical data in which R92L and R92W exhibited severe diastolic impairments at the early time point that improved with increasing age. A normal regulation of Ca2+ kinetics in the context of an altered baseline cTnI phosphorylation suggested a pathogenic mechanism at the myofilament level taking precedence for R92L. The quantitation of Ca2+-handling proteins in R92W mice revealed a synergistic compensatory mechanism involving an increased Ser16 and Thr17 phosphorylation of phospholamban, contributing to the temporal onset of improved cellular mechanics and Ca2+ homeostasis. Therefore, independent cTnT mutations in the TNT1 domain result in primary mutation-specific effects and a differential temporal onset of altered myocellular mechanics, Ca2+ kinetics, and Ca2+ homeostasis, complex mechanisms which may contribute to the clinical variability in cTnT-related familial hypertrophic cardiomyopathy mutations.

Altered hemodynamics in transgenic mice harboring mutant tropomyosin linked to hypertrophic cardiomyopathy

2000 ◽  
Vol 279 (5) ◽  
pp. H2414-H2423 ◽  
Author(s):  
Christian C. Evans ◽  
James R. Pena ◽  
Ronald M. Phillips ◽  
Mariappan Muthuchamy ◽  
David F. Wieczorek ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Protein Kinase ◽  
Left Ventricular ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Fiber Bundles ◽  
Papillary Muscles ◽  
Adrenergic Stimulation ◽  
Skinned Fiber Bundles ◽  
Force Relationship ◽  
Kinase A

We used transgenic (TG) mice overexpressing mutant α-tropomyosin [α-Tm(Asp175Asn)], linked to familial hypertrophic cardiomyopathy (FHC), to test the hypothesis that this mutation impairs cardiac function by altering the sensitivity of myofilaments to Ca2+. Left ventricular (LV) pressure was measured in anesthetized nontransgenic (NTG) and TG mice. In control conditions, LV relaxation was 6,970 ± 297 mmHg/s in NTG and 5,624 ± 392 mmHg/s in TG mice ( P < 0.05). During β-adrenergic stimulation, the rate of relaxation increased to 8,411 ± 323 mmHg/s in NTG and to 6,080 ± 413 mmHg/s in TG mice ( P < 0.05). We measured the pCa-force relationship (pCa = −log [Ca2+]) in skinned fiber bundles from LV papillary muscles of NTG and TG hearts. In control conditions, the Ca2+ concentration producing 50% maximal force (pCa50) was 5.77 ± 0.02 in NTG and 5.84 ± 0.01 in TG myofilament bundles ( P < 0.05). After protein kinase A-dependent phosphorylation, the pCa50 was 5.71 ± 0.01 in NTG and 5.77 ± 0.02 in TG myofilament bundles ( P < 0.05). Our results indicate that mutant α-Tm(Asp175Asn) increases myofilament Ca2+-sensitivity, which results in decreased relaxation rate and blunted response to β-adrenergic stimulation.

Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle

1989 ◽  
Vol 256 (1) ◽  
pp. C96-C100 ◽  
Author(s):  
P. H. Ratz ◽  
C. M. Hai ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Phorbol Esters ◽  
Receptor Activation ◽  
Bay K 8644 ◽  
Cross Bridge ◽  
Cross Bridges ◽  
State Stress

Cross-bridge phosphorylation associated with agonist-stimulated contraction of vascular smooth muscle is often transiently elevated. Such observations led to the concept that phosphorylation of the 20-kDa myosin regulatory light chain (Mp) was required for initial activation and cross-bridge cycling but might not be necessary for steady-state maintenance of stress in the latch state. The possibility that stress maintenance is not regulated by phosphorylation has received some experimental support in contractions induced by phorbol esters and the calcium channel activator BAY K 8644 in which significant increases in Mp were not detected. Our aim was to test the hypothesis that phosphorylation is both necessary and sufficient for activation and for maintenance of steady-state stress. Activation of swine carotid media using agents that bypass receptor activation and elevate Ca2+ influx without mobilizing intracellular Ca2+ stores (BAY K 8644 and ionomycin) produced monotonic increases in both stress and Mp. Transient initial peaks in Mp were absent. Steady-state stress induced by both receptor- and nonreceptor-mediated activation was dependent on small increases in Mp. Increases in Mp greater than 0.3 mol Pi/mol myosin light chain had small effects on stress but produced large increases in the maximum rate of cross-bridge cycling at zero load (Vo). The experimentally determined dependence of stress on Mp was quantitatively predicted by our working hypothesis. This model proposes that Ca2+-stimulated cross-bridge phosphorylation is obligatory for cross-bridge attachment. However, dephosphorylation of attached cross bridges to form noncycling "latch bridges" allows stress maintenance with reduced Mp and cycling.

scholarly journals Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

2015 ◽  
Vol 112 (15) ◽  
pp. 4660-4665 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
David D. Thomas
Keyword(s):  
Light Chain ◽  
Contractile Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Efficiency ◽  
Power Stroke ◽  
Time Resolved ◽  
Muscle Disorders ◽  
Essential Light Chain ◽  
C Terminus

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.

scholarly journals Deletion of 1- 43 Amino Acids from the N-terminus of Myosin Essential Light Chain in Transgenic Mice Decreases Force Production by Reducing the Number of Myosin Cross-Bridges

2011 ◽  
Vol 100 (3) ◽  
pp. 126a
Author(s):  
John J. Michael ◽  
Steven J. Ford ◽  
Katarzyna Kazmierczak ◽  
Danuta Szczesna-Cordary ◽  
Chandra Murali
Keyword(s):  
Amino Acids ◽  
Transgenic Mice ◽  
Light Chain ◽  
Force Production ◽  
Essential Light Chain ◽  
N Terminus ◽  
Cross Bridges

scholarly journals Effects of nicotine administration in a mouse model of familial hypertrophic cardiomyopathy, α-tropomyosin D175N

2011 ◽  
Vol 301 (4) ◽  
pp. H1646-H1655 ◽  
Author(s):  
Robert D. Gaffin ◽  
Shamim A. K. Chowdhury ◽  
Marco S. L. Alves ◽  
Fernando A. L. Dias ◽  
Cibele T. D. Ribeiro ◽  
...  
Keyword(s):  
Heart Rate ◽  
Hypertrophic Cardiomyopathy ◽  
Mouse Model ◽  
Troponin I ◽  
Left Ventricular ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Maximal Rate ◽  
Western Blots ◽  
Nicotine Administration ◽  
Acute And Chronic Effects

The effects of nicotine (NIC) on normal hearts are fairly well established, yet its effects on hearts displaying familial hypertrophic cardiomyopathy have not been tested. We studied both the acute and chronic effects of NIC on a transgenic (TG) mouse model of FHC caused by a mutation in α-tropomyosin (Tm; i.e., α-Tm D175N TG, or Tm175). For acute effects, intravenously injected NIC increased heart rate, left ventricular (LV) pressure, and the maximal rate of LV pressure increase (+dP/d t) in non-TG (NTG) and Tm175 mice; however, Tm175 showed a significantly smaller increase in the maximal rate of LV pressure decrease (−dP/ dt) compared with NTGs. Western blots revealed phosphorylation of phospholamban Ser16 and Thr17 residue increased in NTG mice following NIC injection but not in Tm175 mice. In contrast, phosphorylation of troponin I at serine residues 23 and 24 increased equally in both NTG and Tm175. Thus the attenuated increase in relaxation in Tm175 mice following acute NIC appears to result primarily from attenuated phospholamban phosphorylation. Chronic NIC administration (equivalent to smoking 2 packs of cigarettes/day for 4 mo) also increased +dP/dt in NTG and Tm175 mice compared with chronic saline. However, chronic NIC had little effect on heart rate, LV pressure, −dP/d t, LV wall and chamber dimensions, or collagen content for either group of mice.

scholarly journals Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

2013 ◽  
Vol 304 (2) ◽  
pp. H253-H259 ◽  
Author(s):  
John Jeshurun Michael ◽  
Sampath K. Gollapudi ◽  
Steven J. Ford ◽  
Katarzyna Kazmierczak ◽  
Danuta Szczesna-Cordary ◽  
...  
Keyword(s):  
Amino Acids ◽  
Light Chain ◽  
Sarcomere Length ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Essential Light Chain ◽  
Cross Bridge ◽  
Maximal Activation ◽  
Cardiac Muscle Fibers

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca2+-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa50 (−log10 [Ca2+]free required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa50 values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca2+ sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca2+ sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca2+ sensitivity. Our data suggest that the NH2-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca2+ sensitivity.

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