Abstract 1467: Left Ventricular Function Is Depressed In Mice Expressing Constitutively Pseudo-phosphorylated Cardiac Troponin I
There are three sites on cardiac troponin I (cTnI) that are known to be phosphorylated by protein kinase C (PKC): S43, S45, T144. Several studies have examined the role of PKC-mediated cTnI phosphorylation in cardiac contractile function by mutating these sites to a non-phosphorylatable state. We have created a new transgenic (TG) mouse model (FVB strain) which expresses a mutant cTnI that is pseudo-phosphorylated at the three PKC phosphorylation sites by replacing the endogenous residues with glutamic acid. TG mice were healthy and showed no differences in body mass, left ventricular mass, or myocardial histology as compared to wild-type (WT) mice. Studies were conducted in perfused isolated mouse hearts (37 oC, 1.8 mM extracellular Ca2+) to characterize left ventricular (LV) mechanical function. The slope of the developed pressure-volume relationship was reduced in TG mice (WT: 74.2 ± 3.9 mmHg/ml, n = 6, TG: 64.8 ± 2.5 mmHg/ml, n = 6, P = 0.042), indicating a 13% reduction in LV contractile state. Additionally, TG mice exhibited slowed relaxation as evidenced by a parallel, upward shift of the developed stress-relaxation time relationship (intercept values: WT: 0.025 ± 0.001 ms, n = 6, TG: 0.031 ± 0.002 ms, n = 6, P = 0.004). Responses to isoproterenol treatment (1 μM) were not different between the two groups: developed pressure increased by 40 ± 17 mmHg for WT and 56 ± 17 mmHg for TG (positive inotropy) and relaxation time decreased by 10 ± 6 ms for WT and 13 ± 5 ms for TG (positive lusitropy). PKC-mediated phosphorylation of cTnI is known to be cardioprotective during reperfusion following ischemia. However, TG mice showed no differences with respect to WT mice during ischemia-reperfusion experiments (e.g., fold increase in diastolic pressure from baseline to reperfusion, WT: 4.6 ± 2.3, n = 9, TG: 3.9 ± 1.5, n = 11, P = 0.32), suggesting that the degree of PKC-induced cTnI phosphorylation during reperfusion in WT mice was similar to that of TG mice. In summary, the new transgenic mouse model of constitutively pseudo-phosphorylated cTnI has confirmed that PKC phosphorylation of cTnI has negative ionotropic and lusitropic effects. In addition, the preserved response to isoproterenol suggests that the effects of PKA-mediated and PKC-mediated phosphorylation may be independent.