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Phosphorylation of the troponin inhibitory subunit (TnI) represents a fundamental mechanism of the heart to modulate cardiac contractile function in response to altered physiological or pathological demand. The phosphorylation of TnI at tyrosine 26 (Tyr-26) represents the first myofilament tyrosine phosphorylation identified in the human heart. We demonstrate the phosphorylation of Tyr-26 decreases calcium sensitive force development and accelerates calcium dissociation from the thin filament supporting the role for Tyr-26 phosphorylation in contractile regulation. Additionally, TnI Tyr-26 and the basally phosphorylated Ser-23/24 exhibit functional and signaling integration. That is, the combination of TnI Tyr-26 with Ser-23/24 phosphorylation further accelerates calcium dissociation beyond that of Ser-23/24 or Tyr-26 alone, without additionally decreasing calcium sensitivity. Furthermore, the presence of Ser-23/24 phosphorylation promotes Src family tyrosine kinase mediated phosphorylation of TnI Tyr-26. Taken as a whole, these data support tyrosine kinase mediated myofilament phosphorylation of TnI as a mechanism of cardiac function modulation.

We have engineered cardiac TnCs with increased (L48Q) or decreased (D73N) Ca2+ sensitivity. To express these proteins in the in vivo heart we utilized an adeno-associated virus serotype 9 (AAV-9). The Ca2+ desensitized D73N TnC recapitulated a dilated cardiomyopathy phenotype and depressed function as observed by echocardiography and isolated cardiomyocytes. On the other hand, AAV-9 containing the Ca2+ sensitized L48Q TnC did not cause any disease phenotype or arrhythmias commonly associated with increased myofilament Ca2+ sensitivity. In healthy mice, L48Q TnC increased myocyte contraction and whole heart contractility with improved cardiovascular performance (increased V02max). Excitingly, L48Q TnC expressing mice were able to preserve higher contractility, ejection fraction, cardiac performance and decreased death rate even after undergoing trans-aortic constriction or myocardial infarction. Additionally, L48Q TnC was able to increase contractility, ejection fraction and cardiac performance in mice which expressed L48Q TnC after having a myocardial infarction. In summary, engineered TnCs show potential to be used as treatment strategies against different cardiomyopathies.

Calcium binding to and dissociation from cardiac troponin C (TnC) are essential to cardiac muscle contraction/relaxation. It is well documented that TnC's calcium-binding properties are not constant, but are sensitive to complex interactions between the additional thin and thick filament proteins. There is a growing body of evidence that protein modifications/mutations within different subunits of the Tn complex (TnT, TnI) exert their effects by altering the apparent TnC calcium sensitivity/exchange kinetics. There are a number of potential mechanisms that could alter TnC's calcium-binding, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. We have developed a mathematical model that can simulate the steady-state and kinetic calcium-binding properties of several disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We propose that roughly half of the studied modifications do not alter any of the intrinsic calcium or TnI-binding constants of TnC, but rather the ability of TnC to “find” TnI in the presence of calcium. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium and that TnI also binds to actin, we suggest that the apparent calcium-binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC, which is modulated by Tn modifications. Futhermore, studies with TnC proteins and TnI peptides support the conclusions that TnI concentration strongly influences TnC's calcium-binding properties. Additionally, TnC-TnI chimeras display calcium-binding properties similar to that of TnC in the presence of a saturating TnI concentration. These studies greatly demonstrate the role of TnI concentration in modulating TnC's calcium-binding properties.

Alteration of the Ca2+ binding properties of the thin filament has been implicated as the initiating factor for numerous cardiomyopathies. To directly test if changes in the Ca2+ sensitivity of TnC can lead to altered heart function and a diseased phenotype we utilized specifically engineered cardiac TnCs with modified Ca sensitivities. The specifically designed TnCs allowed us to test whether a direct increase (L48Q TnC) or decrease (D73N TnC) in Ca2+ sensitivity of the myofilament would lead to a hypertrophic or dilated cardiomyopathy, respectively. We used adeno-associated virus serotype 9 (AAV-9) containing either Control TnC, D73N TnC, or L48Q TnC to target the heart. AAV-9 containing GFP or mCherry was used to verify that the AAV-9 was able to infect the heart. The Ca2+ desensitized D73N TnC recapitulated a dilated cardiomyopathy. The hearts presented with dilated ventricles and had enlarged isolated cardiomyocytes that displayed decreased contractility. In addition, nearly half of the D73N mice died suddenly by 8 weeks of age and demonstrated altered electrical properties via ECG - a common feature of human dilated cardiomyopathies. On the other hand, AAV-9 containing the Ca2+ sensitized L48Q TnC did not recapitulate the restrictive or hypertrophic cardiomyopathy phenotypes commonly associated with increased myofilament Ca2+ sensitivity. However, consistent with the enhanced Ca2+ binding abilities of the L48Q TnC, the isolated L48Q TnC cardiomyocytes showed increased contractility, which was recapitulated in vivo via pressure-volume analysis. In summary, the results showed that an alteration in the Ca2+ sensitivity of the myofilament can, but does not always, lead to a diseased heart. In fact these engineered TnCs may be used as a treatment strategy against various cardiac diseases such as myocardial infarction.

Calmodulin (CaM) is known to bind and regulate hundreds of different proteins in a calcium dependent manner. Mammals encode a single isoform of CaM, while plants encode up to ten different isoforms (and dozens of calmodulin-like proteins). Moreover, recent studies have demonstrated that certain soybean CaMs have the ability to differentially activate several mammalian target enzymes in a competitive manner. As a first step in understanding the functional differences among these CaMs, we have studied the Ca2+ and Mg2+ binding properties of two unique soybean CaMs, sCaM1 and sCaM4. CaM has both N-terminal and C-terminal pairs of Ca2+ binding domains (EF-hands). In order to follow cation binding and dissociation from these sCaMs we utilized various fluorescent probes (Trp, Tyr, bis-ANS and quin-2). For each sCaM, our analysis revealed that the rate of Ca2+ dissociation was similar to the regional structural change occurring at each domain, which in turn was similar to the rate of closure of each domain's hydrophobic pocket. Compared to sCaM4, sCaM1 displayed a ∼2-fold lower affinity for Ca2+ at each domain. Consistent with this finding, the N- and C-terminal domains of sCaM1 had ∼5- and 2-fold faster calcium dissociation rates than the respective domains of SCaM4. Both sCaM's N-terminal domain bound Mg2+ with a physiologically relevant affinity (≤1mM), whereas their C-terminal domains had non-physiological Mg2+ binding affinities (≥6mM). The binding of Mg2+ to the N-terminal domains of the SCaMs kept the domain in the closed state and drastically delayed the rate of Ca2+ association and opening of the hydrophobic pocket. Thus, in addition to their ability to differentially regulate enzymes, these soybean CaMs also sense and respond to Ca2+ differently.