Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

We investigated the mechanism by which caffeine influences myofilament responsiveness to Ca2+ by measuring isometric force, Ca2+ binding, and ATPase activity of dog cardiac myofilament proteins. Caffeine (20 mM) increased submaximal and depressed maximal force in skinned fiber bundles. Although the Ca2+ sensitivity of myofilament activity was increased by caffeine, there was no effect on Ca2+ binding to troponin C (TnC) in skinned fiber bundles. To determine if caffeine altered actin-myosin interaction or affected myosin directly, myofibrillar, actomyosin, and myosin ATPase activities were measured. Maximal Ca(2+)-activated myofibrillar Mg(2+)-ATPase activity was depressed by 20 mM caffeine, whereas submaximal Mg(2+)-ATPase activities were not changed. Actomyosin Mg(2+)-ATPase activity was significantly depressed by caffeine concentrations > or = 15 mM. Myosin Ca(2+)-ATPase activity was depressed by caffeine, whereas Mg(2+)-ATPase and K(EDTA)-ATPase activities were not affected. These data suggest that caffeine affects myofilament function via a mechanism that is independent of TnC-Ca2+ binding but that may involve direct effects on actin-cross-bridge interaction.

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PKC catalytic subunit and phenylephrine induce a comparable decrease in myofilament calcium sensitivity

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2007.03.129 ◽

2007 ◽

Vol 42 (6) ◽

pp. S46

Author(s):

Judit Barta ◽

Jolanda van der Velden ◽

Nicky M. Boontje ◽

Ruud Zaremba ◽

Ger J.M. Stienen

Keyword(s):

Calcium Sensitivity ◽

Catalytic Subunit ◽

Myofilament Calcium Sensitivity

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Abstract 219: Histidine Substituted Cardiac Troponin I (a164h) Improves Survival And Protects Cardiac Contractility During Acute Hypoxia In The Aged Murine Heart.

Circulation ◽

10.1161/circ.116.suppl_16.ii_23-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Nathan Palpant ◽

Sharlene Day ◽

Kimber Converso ◽

Joseph Metzger

Keyword(s):

Cardiac Troponin ◽

Troponin I ◽

Acute Hypoxia ◽

Cardiac Contractility ◽

Systolic Pressure ◽

Calcium Sensitivity ◽

The Elderly ◽

Cardiac Troponin I ◽

Pump Failure ◽

Myofilament Calcium Sensitivity

Contractile dysfunction associated with ischemia is a significant cause of morbidity and mortality particularly in the elderly. Strategies designed to protect the aged heart from ischemia-mediated pump failure are needed. We have generated transgenic (Tg) mice expressing a modified form of adult cardiac troponin I, the Ca ++ -activated molecular switch of the myofilament. Consonant with the fetal isoform, this transgene encodes a histidine substitution (A164H) in the critical switch domain of TnI thus increasing myofilament calcium sensitivity in a pH-dependent manner. We hypothesized that aged mice (24 months), intrinsically susceptible to myocardial dysfunction, would retain improved cardiac contractility at baseline and during an acute hypoxic challenge by means of myofilament-mediated calcium sensitization. Methods/Results: At baseline, by echocardiography, Tg hearts had increased systolic function, with a 26% higher mean ejection fraction compared to nontransgenic (Ntg) mice: 75 ± 3% [Tg: n = 13] vs. 63 ± 4% [Ntg: n = 12], P < 0.05, with no differences in diastolic function between the groups. To study the effects of acute hypoxia on cardiac hemodynamics mice underwent microconductance Millar catheterization while ventilated with 12% oxygen. Aged Tg mice had improved survival compared to Ntg mice: time to pump failure (65% of baseline peak systolic pressure) 11.59 ± 1.25 min. [Tg: n = 3] vs. 4.11 ± 1.90 min. [Ntg: n = 3], P < 0.05. After four minutes of hypoxia, Tg mice had markedly improved cardiac contractility compared to Ntg mice with increased stroke volume (30.05 ± 4.49 uL [Tg] vs. 13.23 ± 3.21 uL [Ntg], P < 0.05), end systolic pressure (106.09 ± 11.81 mmHg [Tg] vs. 64.49 ± 4.05 mmHg [Ntg], P < 0.05) and rate of positive left ventricular pressure development (12958.66 ± 2544.68 mmHg/sec [Tg] vs. 5717.00 ± 745.67 mmHg/sec [Ntg], P = 0.05). Conclusion: An alteration in myofilament calcium sensitivity via a pH-responsive histidine button in cardiac troponin I augments baseline heart function in Tg mice over their lifetime. During acute hypoxia, cTnI A164H improves survival in aged mice by maintaining cardiac contractility, and thus holds promise for the design of gene therapeutics to treat pump failure associated with acute ischemic events in the elderly.

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High-throughput assessment of calcium sensitivity in skinned cardiac myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.2.h969 ◽

2001 ◽

Vol 281 (2) ◽

pp. H969-H974 ◽

Cited By ~ 18

Author(s):

Chee Chew Lim ◽

Michiel H. B. Helmes ◽

Douglas B. Sawyer ◽

Mohit Jain ◽

Ronglih Liao

Keyword(s):

Cardiac Myocytes ◽

High Throughput ◽

High Throughput Screening ◽

Rapid Determination ◽

Isometric Force ◽

Calcium Sensitivity ◽

Pharmacological Agents ◽

Length Changes ◽

Myofilament Calcium Sensitivity ◽

High Degree

Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.

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Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00134.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. H548-H556 ◽

Cited By ~ 6

Author(s):

Renan J. Sandoval ◽

Elisha R. Injeti ◽

James M. Williams ◽

William T. Georthoffer ◽

William J. Pearce

Keyword(s):

Calcium Influx ◽

Cerebral Arteries ◽

Calcium Sensitivity ◽

Cytosolic Calcium ◽

Stretch Ratio ◽

Myogenic Tone ◽

Postnatal Maturation ◽

Calcium Sensitization ◽

Myofilament Calcium Sensitivity ◽

Krebs Buffer

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K+ (Kmax) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca2+ concentration at stretch ratios >2.0 in adult arteries, but increased Ca2+ concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %Kmax values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 ± 2.3%). Conversely, adult %Kmax values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 ± 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.

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