Measurement of Calcium Dissociation Rates from Troponin C in Rigor Skeletal Myofibrils

Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca2+ dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca2+ dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin CIAEDANS, reported Ca2+ dissociation exclusively from the structural domain of troponin C at ∼0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca2+ dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin CMIANST54C,C101S reported Ca2+ dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin CIAANST54C,C101S reported a biphasic fluorescence change upon Ca2+ dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin CMIANST54C,C101S and troponin CIAEDANS at all levels of the troponin C systems. Furthermore, the rate of Ca2+ dissociation from troponin C in the myofibrils was similar to the rate of Ca2+ dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca2+ dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca2+ dissociation from troponin C may influence relaxation

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin-Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral-mediated delivery to alleviate arrhythmias in non-CaM-related CPVT

Disease-Related Cardiac Troponins Alter Thin Filament Ca2+ Association and Dissociation Rates

The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C, , we examined the effects of ten disease-related troponin modifications on the Ca2+ binding properties of the troponin complex and the reconstituted thin filament. The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies (dilated, hypertrophic and restrictive) and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca2+ binding properties of the isolated troponin complex. However, when incorporated into the thin filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the thin filament to Ca2+ (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca2+ dissociation from the thin filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca2+ association to the thin filament. Thus, the disease-related protein modifications alter Ca2+ binding by influencing both the association and dissociation rates of thin filament Ca2+ exchange. These alterations in Ca2+ exchange kinetics influenced the response of the thin filament to artificial Ca2+ transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca2+-binding properties of the thin filament and influence the contractile performance of the heart

Myofilament Calcium Sensitivity: Consequences of the Effective Concentration of Troponin I

Control of calcium binding to and dissociation from cardiac troponin C (TnC) is essential to healthy cardiac muscle contraction/relaxation. There are numerous aberrant post-translational modifications and mutations within a plethora of contractile, and even non-contractile, proteins that appear to imbalance this delicate relationship. The direction and extent of the resulting change in calcium sensitivity is thought to drive the heart toward one type of disease or another. There are a number of molecular mechanisms that may be responsible for the altered calcium binding properties of TnC, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium, we suggest that the apparent calcium binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC. Based on our previous work, TnI peptide binding studies and the calcium binding properties of chimeric TnC-TnI fusion constructs, and building upon the concept of effective concentration, we have developed a mathematical model that can simulate the steady-state and kinetic calcium binding properties of a wide assortment of disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We predict that several TnI and TnT modifications do not alter any of the intrinsic calcium or TnI binding constants of TnC, but rather alter the ability of TnC to “find” TnI in the presence of calcium. These studies demonstrate the apparent consequences of the effective TnI concentration in modulating the calcium binding properties of TnC

The relationship between changes in the Ca²⁺ sensitivity and the rate of Ca²⁺ dissociation.

<p>The changes in the thin filament Ca²⁺ sensitivity for the ten disease-related protein modifications are plotted against the changes in the rate of Ca²⁺ dissociation from the thin filament. The straight line in the figure represents a perfect correlation between the thin filament change in Ca²⁺ sensitivity and Ca²⁺ dissociation rate.</p