John S. Walker

The Troponin C G159D Mutation Blunts Myofilament Desensitization Induced by Troponin I Ser23/24 Phosphorylation

Striated muscle contraction is regulated by the binding of Ca2+ to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, &bgr;-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca2+-dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca2+ in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca2+ sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca2+-sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca2+-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca2+-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through &bgr;-adrenergic signaling as well as the development of cardiomyopathy.

Biochemical and myofilament responses of the right ventricle to severe pulmonary hypertension.

Right ventricular (RV) failure is one of the strongest predictors of mortality both in the presence of left ventricular decompensation and in the context of pulmonary vascular disease. Despite this, there is a limited understanding of the biochemical and mechanical characteristics of the pressure-overloaded RV at the level of the cardiac myocyte. To better understand this, we studied ventricular muscle obtained from neonatal calves that were subjected to hypobaric atmospheric conditions, which result in profound pulmonary hypertension. We found that RV pressure overload resulted in significant changes in the phosphorylation of key contractile proteins. Total phosphorylation of troponin I was decreased with pressure overload, predominantly reflecting changes at the putative PKA site at Ser(22/23). Similarly, both troponin T and myosin light chain 2 showed a significant decline in phosphorylation. Desmin was unchanged, and myosin-binding protein C (MyBP-C) phosphorylation was apparently increased. However, the apparent increase in MyBP-C phosphorylation was not due to phosphorylation but rather to an increase in MyBP-C total protein. Importantly, these findings were seen in all regions of the RV and were paralleled by reduced Ca(2+) sensitivity with preserved maximal Ca(2+) saturated developed force normalized to cross-sectional area in isolated skinned right ventricular myocyte fragments. No changes in total force or cooperativity were seen. Taken together, these results suggest that RV failure is mechanistically unique from left ventricular failure.

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils.

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.

Titin and the Developing Heart

See related articles, pages 967–975 and Circ Res. 2004;94:505–513 During gestation, the mammalian heart operates under very different loading conditions than those seen during adult life. Ensuring that cardiac output is sufficient at the low filling pressures found in the fetal circulation requires mechanical strategies and protein complements different from those seen in the adult. The studies by Opitz et al,1 in this issue of Circulation Research , and Lahmers et al,2 published recently in this journal, provide some clues as to the molecular basis of these strategies. Both studies examined the developing heart and found correlations between the expression of titin isoforms and the increased stiffness of the myocardium as the organism progressed from fetus through neonate to adult. The pattern of findings is remarkably consistent across both studies and, despite differences with earlier claims of a less compliant fetal heart, leads to the conclusion that the fetal sarcomere is more compliant than the adult and that this is in large part due to the expression of particular isoforms of titin. But why is a more compliant titin isoform beneficial to the fetus? We will briefly review the properties of titin here. Primary sources may be found by consulting recent reviews.3–9 Titin, also known as connectin, is a relatively recently discovered giant protein (3 to 4 MDa ) that is the third most abundant protein in striated muscle, forming up to 10% of the total protein content of the cell. Titin extends half the length of the sarcomere from the Z-disc through the I-band and A-band to the M-line, a distance of about 1 μm.7 The N-terminal end of titin is capped by telethonin, a 19-kDa protein that appears to be confined to striated muscle and that may have a role in organizing myofibrillogenesis.10–12 …