Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca2+ signal. Promisingly, our smartly formulated Ca2+-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.

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.

Troponin I (TnI) is a major regulator of cardiac muscle contraction and relaxation. During physiological and pathological stress, TnI is differentially phosphorylated at multiple residues through different signaling pathways to match cardiac function to demand. The combination of these TnI phosphorylations can exhibit an expected or unexpected functional integration, whereby the function of two phosphorylations are different than that predicted from the combined function of each individual phosphorylation alone. We have shown that TnI Ser-23/24 and Ser-150 phosphorylation exhibit functional integration and are simultaneously increased in response to cardiac stress. In the current study, we investigated the functional integration of TnI Ser-23/24 and Ser-150 to alter cardiac contraction. We hypothesized that Ser-23/24 and Ser-150 phosphorylation each utilize distinct molecular mechanisms to alter the TnI binding affinity within the thin filament. Mathematical modeling predicts that Ser-23/24 and Ser-150 phosphorylation affect different TnI affinities within the thin filament to distinctly alter the Ca2+-binding properties of troponin. Protein binding experiments validate this assertion by demonstrating pseudo-phosphorylated Ser-150 decreases the affinity of isolated TnI for actin, whereas Ser-23/24 pseudo-phosphorylation is not different from unphosphorylated. Thus, our data supports that TnI Ser-23/24 affects TnI-TnC binding, while Ser-150 phosphorylation alters TnI-actin binding. By measuring force development in troponin-exchanged skinned myocytes, we demonstrate that the Ca2+ sensitivity of force is directly related to the amount of phosphate present on TnI. Furthermore, we demonstrate that Ser-150 pseudo-phosphorylation blunts Ser-23/24-mediated decreased Ca2+-sensitive force development whether on the same or different TnI molecule. Therefore, TnI phosphorylations can integrate across troponins along the myofilament. These data demonstrate that TnI Ser-23/24 and Ser-150 phosphorylation regulates muscle contraction in part by modulating different TnI interactions in the thin filament and it is the combination of these differential mechanisms that provides understanding of their functional integration.

Troponin I (TnI), the inhibitory subunit of the troponin complex, can be phosphorylated as a key regulatory mechanism to alter the calcium regulation of contraction. Recent work has identified phosphorylation of TnI Tyr-26 in the human heart with unknown functional effects. We hypothesized that TnI Tyr-26 N-terminal phosphorylation decreases calcium sensitivity of the thin filament, similar to the desensitizing effects of TnI Ser-23/24 phosphorylation. Our results demonstrate Tyr-26 phosphorylation and pseudo-phosphorylation decrease calcium binding to Troponin C (TnC) on the thin filament and calcium sensitivity of force development to a similar magnitude as TnI Ser-23/24 pseudo-phosphorylation. To investigate the effects of TnI Tyr-26 phosphorylation on myofilament deactivation, we measured the rate of calcium dissociation from TnC. Results demonstrate filaments containing Tyr-26 pseudo-phosphorylated TnI accelerate the rate of calcium dissociation from TnC similar to that of TnI Ser-23/24. Finally, to assess functional integration of TnI Tyr-26 with Ser-23/24 phosphorylation, we generated recombinant TnI phospho-mimetic substitutions at all three residues. Our biochemical analyses demonstrated no additive effect on calcium sensitivity or calcium-sensitive force development imposed by Tyr-26 and Ser-23/24 phosphorylation integration. However, integration of Tyr-26 phosphorylation with pseudo-phosphorylated Ser-23/24 further accelerated thin filament deactivation. Our findings suggest that TnI Tyr-26 phosphorylation functions similarly to Ser-23/24 N-terminal phosphorylation to decrease myofilament calcium sensitivity and accelerate myofilament relaxation. Furthermore, Tyr-26 phosphorylation can buffer the desensitization of Ser-23/24 phosphorylation while further accelerating thin filament deactivation. Therefore, the functional integration of TnI phosphorylation may be a common mechanism to modulate Ser-23/24 phosphorylation function.