An internal domain of β-tropomyosin increases myofilament Ca²⁺sensitivity

Abstract

-mediated muscle contraction and relaxation in the heart. Striated muscle ␣-TM is the major isoform expressed in the heart. The expression of striated muscle ␤-TM in the murine myocardium results in a decreased rate of relaxation and increased myofilament Ca 2ϩ sensitivity. Replacing the carboxyl terminus (amino acids 258 -284) of ␣-TM with ␤-TM (a troponin T-binding region) results in decreased rates of contraction and relaxation in the heart and decreased myofilament Ca 2ϩ sensitivity. We hypothesized that the putative internal troponin T-binding domain (amino acids 175-190) of ␤-TM may be responsible for the increased myofilament Ca 2ϩ sensitivity observed when the entire ␤-TM is expressed in the heart. To test this hypothesis, we generated transgenic mice that expressed chimeric TM containing ␤-TM amino acids 175-190 in the backbone of ␣-TM (amino acids 1-174 and 191-284). These mice expressed 16 -57% chimeric TM and did not develop cardiac hypertrophy or any other morphological changes. Physiological analysis showed that these hearts exhibited decreased rates of contraction and relaxation and a positive response to isoproterenol. Skinned fiber bundle analyses showed a significant increase in myofilament Ca 2ϩ sensitivity. Biophysical experiments demonstrated that the exchanged amino acids did not influence the flexibility of the TM. This is the first study to demonstrate that a specific domain within TM can increase the Ca 2ϩ sensitivity of the thin filament and affect sarcomeric performance. Furthermore, these results enhance the understanding of why TM mutations associated with familial hypertrophic cardiomyopathy demonstrate increased myofilament sensitivity to Ca 2ϩ .calcium sensitivity; contractile function; genetically altered mice TROPOMYOSIN (TM) is an essential sarcomeric protein that plays an integral role in the regulation of Ca 2ϩ -mediated striated muscle contraction. Through its interactions with the troponin (Tn) complex, TM shifts its conformational position on actin in response to Ca 2ϩ binding to TnC; this repositioning of TM on actin exposes the myosin-binding site on actin and facilitates muscle contraction. Upon cessation of stimulation, there is a resequestration of Ca 2ϩ into the sarcoplasmic reticulum and extrusion of Ca 2ϩ from the cell, resulting in TM returning to its original position as the muscle fiber relaxes.Previous studies (18,24,25) in our laboratory have established there are physiological differences among the three highly conserved striated muscle TM isoforms. The ␣-TM isoform is the predominant isoform in both skeletal and cardiac musculature in the mouse, constituting ϳ98% of total TM protein in the heart. Transgenic (TG) mice that express 50 -60% ␤-TM protein in the heart demonstrate increased sensitivity of the cardiac myofilaments to Ca 2ϩ , which leads to diastolic dysfunction (18). When ␤-TM is expressed at high levels (75-80% ␤-TM protein) in the heart, mice die due to defects in both contraction and relaxation (17). Overexpression of wild-type ␣-TM does not lea...

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin

“…Differences in Ca 2+ -sensitivity or cooperativity of force generation of striated muscle are associated with different isoforms of Tm and TnT (e.g. Schachat et al, 1987;Greaser et al, 1988;Palmiter et al, 1996;Gallon et al, 2006;McCall et al, 2006;Jagatheesan et al, 2009). It is possible, therefore, that significant differences in the Ca 2+ regulation of force generation between jaw-closing and limb muscles within individual species and in jaw-closing muscles between species are associated with the differences in the Tm and TnT isoform expression patterns observed in this study.…”

“…Different isoforms of TnT and Tm are associated with variations in the Ca 2+ -sensitivity or cooperativity of force generation in muscle, as determined by shifts in the force- [Ca 2+ ] relationship in fibers in which the surface membrane is removed or rendered hyperpermeable (e.g. Schachat et al, 1987;Greaser et al, 1988;Palmiter et al, 1996;Ogut et al, 1999;McCall et al, 2006;Biesiadecki et al, 2007;Jagatheesan et al, 2009). Furthermore, Tm isoforms can differentially modulate contractile properties through the TnT-binding domain, independent of differences in Ca 2+ -sensitivity (Jagatheesan et al, 2004).…”

Striated muscle tropomyosin isoforms differentially regulate cardiac performance and myofilament calcium sensitivity

“…Age matched (4-month-old) NTG control, β -TM TG, γ -TM TG and βγ -DTG mice were subject to the work performing heart analysis as described (Jagatheesan et al 2007, 2009). Data are presented as means ± SEM.…”

“…We measured force developed by bundles of detergent-extracted fibers obtained from 4-month-old papillary muscle as described (Jagatheesan et al 2003, 2004, 2009; Wolska et al 1999). Data in which measurements were made in the same fiber bundle at different sarcomere lengths were analyzed individually using Student’s paired t -test.…”

“…Interactions of TM with actin and/or Tn may be involved in these differential functions. Previous studies employing chimeric TM molecules clearly established that the putative internal and carboxyl terminal TnT binding domains of α - and β -TM impart different functions with respect to myocardial performance (Jagatheesan et al 2003, 2004, 2009). The physiological parameters that are most dramatically affected are associated with the rates of contraction and relaxation, time to peak left ventricular pressure development, duration of time for left ventricular relaxation, and calcium sensitivity of the myofilament.…”