Development and Cardiac Contractility: Cardiac Troponin T Isoforms and Cytosolic Calcium in Rabbit

Abstract

Cardiac contractility depends on calcium sensitivity of the myofilaments and cytosolic free calcium concentration ([Ca 2ϩ ] i ) during activation. During development, the cardiac troponin T isoform cTnT 1 is replaced by shorter cTnT isoforms, including cTnT 4 , and changes occur in other myofibrillar proteins and in calcium regulation. We expressed rabbit recombinant (r)cTnT 1 and rcTnT 4 in Spodoptera frugiperda cells and determined their effect on calcium binding to TnC in solution and on the calcium sensitivity of myofilaments in skinned rabbit ventricular fibers in vitro. We measured [Ca 2ϩ ] i and L-type calcium current (I Ca ) in ventricular myocytes from 3-wk-old and adult rabbits. Adult myocardium develops more force than fetal myocardium (1). Adult cardiac myocytes shorten further and faster than immature myocytes (2). Potential contributors to these differences include developmental increases in calcium ([Ca 2ϩ ] i ) transients and calcium sensitivity of myofilaments (1,3). Calcium transients increase postnatally in the rabbit (4). The calcium sensitivity of myofilaments, however, decreases with development in the rat, chick, and rabbit (5-7); this fall could result from a developmental switch in isoform expression of cTnT, a thin filament protein essential for Ca 2ϩ -regulated force development (8). Expression of cTnT 1 and cTnT 2 falls and that of cTnT 3 and cTnT 4 increases with development in the rabbit.We expressed recombinant rabbit (r)cTnT 1 and rcTnT 4 in Sf9 cells and compared their effects on Ca 2ϩ -binding of isolated troponin in solution and in skinned muscle fibers in vitro. Further, we compared calcium transients and L-type calcium currents (I Ca ) in cardiac myocytes from 3-wk-old rabbits expressing cTnT 1 and from adults that express predominantly cTnT 4 and no cTnT 1 .We found that (a) in solution, Tn comprising cTnT 1 (Tn cTnT1 ) has higher calcium affinity than Tn comprising cTnT 4 (Tn cTnT4 ), (b) fibers reconstituted with cTnT 1 were more sensitive to calcium than those reconstituted with cTnT 4 , and (c) peak [Ca 2ϩ ] i and I Ca were significantly greater in adult than in 3-wk-old myocytes. Our data provide no evidence that incorporation of Tn into the myofilaments alters Tn-calcium binding affinity.By imparting to the myofilaments a higher calcium sensitivity, the higher expression of cTnT 1 and cTnT 2 in immature myocytes would compensate partially for their smaller [Ca 2ϩ ] i transients. MATERIALS AND METHODSAnimals used in this study were handled according to the animal welfare regulations of Duke University (Durham, NC). The protocol was approved by the Institutional Animal Care and Use Committee. The rabbits were anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine i.p.), and the heart was removed to obtain ventricular muscle fibers or isolated myocytes.Preparation of cTnT. cTnT isoforms were expressed in Sf9 cells using the baculovirus system (Invitrogen). The cDNA of rabbit cTnT 1 (9) was cloned at the EcoR I restriction site into pVL1392 by releas...

Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness

“…S17-S19). The intracellular tension and contractility of primary CMs also depend on these intra-and extrasarcomeric cytoskeletal structures (1,31,36,39). The expression levels of titin isoforms (N2A and N2B) and TnI and TnT differed in cultures of 7:1 hPSC-CMs on polyacrylamide vs. glass (SI Appendix, Fig.…”

“…We next examined expression of titin, which regulates tension along myofibrils (36,37), and troponin I (TnI) and troponin T (TnT), which regulate sarcomere contraction (38,39). We compared expression of TnI, TnT, and titin isoforms (N2A and N2B) in unpatterned hPSC-CMs on 10-kPa hydrogels vs. patterned on glass and 10-and 35-kPa hydrogels (SI Appendix, Fig.…”

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).…”

The influence of trout cardiac troponin I and PKA phosphorylation on the Ca2+ affinity of the cardiac troponin complex

“…To more completely characterize the influence of trout cTnI on the Ca 2+ affinity of the Tn complex, further work needs to be completed with this protein complexed with trout cTnC and trout cTnT. Previous work has illustrated that each of the troponin components influences the Ca 2+ binding characteristics of the Tn complex (Abbott et al, 2001;Davis et al, 2007;Liang et al, 2008;McAuliffe et al, 1990;McCall et al, 2006;Robertson et al, 1982). It may be, therefore, that all trout components need to be working together for the full effect of the functional characteristics of the proteins to be realized.…”