Dilated cardiomyopathy (DCM) is a disease of cardiac muscle characterized by left ventricular dilation and systolic dysfunction. DCM is believed to be caused by mutations in cytoskeletal and sarcomeric proteins in 30- 40 % of all cases. Understanding how these mutations lead to the development of DCM is lacking, although most known DCM-linked mutations of sarcomeric proteins desensitize cardiac muscle to Ca2+. However, the role of Ca2+ sensitivity of cardiac troponin C (cTnC) in triggering the pathogenesis of DCM is a matter of ongoing debate. The objective of the proposed project is to determine whether we can generate a mutation in cTnC not discovered in nature and then introduce it into mouse myocardium to recreate the phenotype of DCM. In order to achieve this objective, we designed a cTnC mutant that led to reduced Ca2+ sensitivity and faster rate of Ca2+ dissociation from reconstituted thin filaments. We then introduced the Ca2+ desensitizing cTnC mutation into mouse myocardium via gene targeting technology, generating a novel Tnnc1 knock-in mouse model. The novel knock-in mouse model will enable us to evaluate the role of desensitizing cTnC to Ca2+ in triggering the pathogenesis of DCM. To evaluate the role of Ca2+ sensitivity of cTnC in the development of DCM, the proposed project will utilize the following aims:
Specific Aim 1 : Examine the effect of Ca2+ desensitizing (including DCM-linked) cTnC mutations on the response of reconstituted thin filaments to Ca2+ upon cTnI phosphorylation.
This aim will test the hypothesis that Ca2+ desensitizing (including DCM-linked) cTnC mutations diminish the effect of phosphorylation of cTnI by protein kinase A (PKA) on Ca2+ binding and exchange with reconstituted thin filaments. We will evaluate the effect of decreasing Ca2+ sensitivity of cTnC on the response of reconstituted thin filaments to Ca2+ upon cTnI phosphorylation by PKA.
Specific Aim 2 : Determine whether decreasing the Ca2+ sensitivity of cTnC leads to pathogenesis of DCM.
This aim will test the hypothesis that decreasing the Ca2+ sensitivity of cTnC (by accelerating the rate of Ca2+ dissociation) leads to the development of DCM. The results from two experimental groups (heterozygous and homozygous knock-in mice) will be compared to those for control group (wild-type mice). The consequences of the D73N cTnC mutation are expected to be more severe for homozygous mice than for heterozygous mice.
Specific Aim 3 : Determine the effect of decreasing the Ca2+ sensitivity of cTnC on contractile function.
This aim will test the hypothesis that decreasing the Ca2+ sensitivity of cTnC (by accelerating the rate of Ca2+ dissociation) leads to systolic dysfunction. Systolic function will be assessed at the whole animal level using echocardiography. Systolic function will also be assessed at the cellular level utilizing ventricular myocytes isolated from the hearts of heterozygous, homozygous and wild- type mice. The proposed research will provide a deeper insight into the molecular mechanism of DCM.
Dilated cardiomyopathy is a disorder of cardiac muscle that is characterized by left ventricular dilation and systolic dysfunction. Understanding whether interfering with normal Ca2+ binding properties of cardiac troponin C triggers the pathogenesis of dilated cardiomyopathy might lead to the rational design of novel treatments for this debilitating disease.
|Liu, Yu; Chen, Li; Diaz, Andrea Diaz et al. (2016) Mesp1 Marked Cardiac Progenitor Cells Repair Infarcted Mouse Hearts. Sci Rep 6:31457|
|Swindle, Nicholas; Albury, Acchia N J; Baroud, Belal et al. (2014) Molecular and functional consequences of mutations in the central helix of cardiac troponin C. Arch Biochem Biophys 548:46-53|