Regulation Of Diastolic Function By NH 2 -terminal Phosphorylation Of Cardiac Troponin I

Cardiac troponin I (cTnI) is a key protein in regulation of cardiac contractility. There are two kinds of mechanisms to modify the cTnI function: phosphorylation and non-phosphorylation. Degradation regulates the cTnI function and cardiac contractility in a non-phosphorylation way, by removing the phosphorylation sites, or changing the allosteric conformation of cTnI.Our previous work found cTnI NH2-terminal degradation (cTnI-ND) markedly increased in the rat myocardium after 4 weeks'tail-suspension. cTnI-ND lacked two protein kinase A (PKA)-targeted phosphorylation sites (Ser23 and Ser24). PKA phosphorylation of cTnI reduces myofilament Ca2+ sensitivity, and increases the rate of Ca2+ dissociation and contributes to an acceleration of relaxation. PKA-dependent cTnI phosphorylation increases the crossbridge cycling rate and enhances the unloaded shortening velocity. We hypothesized that the increased cTnI-ND in tail-suspended rats might affect cardiac function, especially duringβ-adrenergic stimulation. In order to observe the effect of cTnI-ND on cardiac function of tail-suspended rats, we investigated the responsiveness of isolated working heart toβ-adrenergic agonist isoproterenol (ISO), the responsiveness of single cardiomyocyte toβ-adrenergic receptor signaling pathway agonists, and the force-pCa relationship of skinned papillary fibers. Expression and phosphorylation of related proteins were also tested by western blot and immunohistochemisty. The results are as follow:1. Reduced responsiveness of isolated working heart to ISO in tail-suspended rats. Under basal condition there was a trend for decreased cardiac output in tail-suspended rats, while the increase in cardiac output with ISO was lower in tail-suspended rats compared with controls. The left ventricular end-diastolic pressure (LVEDP) and maximal rates of left ventricular pressure relaxation (-dP/dtmax) were lower in tail-suspended rats. And the decrease in LVEDP and increase in -dP/dtmax which were induced by ISO were limited in tail-suspended rats.2. Depressed responsiveness of cardiomyocytes toβ-adrenergic receptor signaling pathway agonists in tail-suspended rats. At stimulation of 1 Hz, 2 Hz and 4 Hz, the shortening amplitude of cardiomyocytes was lower in tail-suspended rats. The responsiveness to ISO, forskolin, DB-cAMP, and IBMX was lower in cardiomyocytes from tail-suspended rats. At maximal dose of ISO, forskolin, DB-cAMP, and IBMX treatment, the increase in shortening and re-lengthening rates and the decrease in shortening and re-lengthening time of cardiomyocytes were limited in tail-suspended rats.3. There was no difference in myofibril Ca2+ sensitivity between tail-suspended and control rats, although myofibril Ca2+ sensitivity was decreased less in tail-suspended versus control rats during PKA phosphorylation.4. Four weeks of tail-suspension increased cTnI-ND and reduced cTnI phosphorylation by ISO treatment.5. There was no difference in PKA protein expression and activation during ISO stimulation between the control and tail-suspended groups. 6. The expression of PLB and phosphorylation at Ser16 by ISO were unchanged in the tail-suspended rats.These data suggest that enhanced cTnI-ND after tail-suspension is an important factor for reduced diastolic function in tail-suspended rat heart, and the limited increase in relaxation rate may reduce cardiac output in tail-suspended rat heart. This may be a factor that causes orthostatic intolerance after astronauts returning to earth.In order to understand the effect of cTnI NH2-terminal and its phosphorylation on diastolic function, we compared the isoproterenol-dependent acceleration of relaxation (IDAR) and the frequency-dependent acceleration of relaxation (FDAR) in isolated working hearts and cardiomyocytes. The results are as follow:1. ISO caused more enhancements in diastolic function of isolated working hearts than electrical pacing. At 20 nM ISO stimulation, the heart rate elevated to approximately 300 beats/min, while LVEDP markedly decreased, and -dP/dtmax increased in working heart. But at 300 beats/min pacing, LVEDP increased and -dP/dtmax decreased in working heart. As for the relaxation time, ISO reduced more than electrical pacing. Under 420 beats/min pacing, the relaxation time reduced most at 10 nM ISO treatment.2. ISO promoted Ca2+ re-uptake, and enhanced systolic and diastolic function in cardiomyocytes. The peak and resting calcium transient increased with stimulation frequency, while ISO increased the peak calcium transient and decreased the resting calcium concentration. Perfusing with ISO, the resting calcium concentration still increased with stimulation, but was much lower than that without ISO. As for relaxation time, ISO decreased more than electrical stimulation.3. Phosphorylation of cTnI at Ser23/24 increased with ISO concentration, but didn't change with electrical pacing.4. ISO increased phosphorylation of PLB at Ser16 and Thr17, but the increase in phosphorylation at Ser16 was more significant. Electrical pacing only increased the phosphorylation at Thr17. While perfusing with ISO, electrical pacing could still increase the phosphorylation at Thr17.These data suggest that electrical pacing increased only phosphorylation of PLB at Thr17, and had less effect on diastolic function. But after ISO treatment, both PLB and cTnI were phosphorylated, and a synergic effect between PLB and cTnI caused further enhancement in the relaxation.