Force relaxation and thin filament protein phosphorylation during acute myocardial ischemia

Abstract

Ischemia impairs myocardial function and may contribute to the progression of heart failure. In this study, rats subjected to acute ischemia demonstrated reduced Ca 2+ activated force as well as a decrease in myosin binding protein-C, titin and Ser23/24 phosphorylation of troponin I (TnI). All three proteins have been demonstrated to be downstream targets of β-adrenergic receptor activation (β-AR), leading to the hypothesis that decreased β-AR during ischemia leads to reduced protein phosphorylation and reduced rate constants of force relaxation. To test this hypothesis, force relaxation transients were recorded from permeabilized perfused and ischemic rat heart fibers following photolysis of the caged chelator diazo-2. Relaxation transients were best fit by double exponential functions whereby the majority (>70%) of the force decline was described by the fast rate constant, which was ~5 times faster than the slow rate constant. However, rate constants of relaxation between perfused and ischemic fibers were not different, despite significant decreases in sarcomeric protein phosphorylation in ischemic fibers. Treatment of perfused fibers with a cAMP analog increased Ser23/24 phosphorylation of TnI, yet the rate constants of relaxation remained unchanged. Interestingly, similar treatment of ischemic fibers did not impact TnI phosphorylation or force relaxation transients. Therefore, acute ischemia does not influence the rate constants of relaxation of permeabilized fibers. These results also suggest that the physiological level of sarcomeric protein phosphorylation is unlikely to be the primary driver of relaxation kinetics in permeabilized cardiac muscle fibers.

The Rates of Ca2+ Dissociation and Cross-bridge Detachment from Ventricular Myofibrils as Reported by a Fluorescent Cardiac Troponin C

“…This result may not be surprising considering that previous studies have shown that myosin S1 and singleheaded myosin have different properties than even heavy meromyosin (55). In agreement with our results, the apparent rates of ADP dissociation measured from the rabbit, rat, and human myofibrils were similar to the calculated rates of ADP dissociation measured by the laser trap assay (rabbit and rat) (41,44) and from the sinusoidal length perturbation analysis of crossbridge detachment (human, rabbit, and rat) (52)(53)(54).…”

“…Previously, the rates of ADP dissociation from different cardiac myosins were inferred from the study of unregulated actomyosin in solution (20), laser trap experiments (44), and sinusoidal analysis on skinned cardiac muscle (52)(53)(54). The apparent rate of ADP dissociation measured by our fluorescent TnC is nearly one order of magnitude slower than that measured from actomyosin in solution for the different fast and slow myosins (20).…”

Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca2+ sensitivity and accelerates deactivation in an acidic environment

“…Conversely, approximately 40% of TnI is basally phosphorylated at both Ser-23 and 24 [9]. While TnI Ser-23/24 was previously reported to be decreased by approximately 30% in the ischemic, non-perfused rat papillary muscle [24, 25], we demonstrate ischemia increases TnI Ser-23/24 phosphorylation by 34% in the mouse left ventricular free wall containing both perfused and ischemic tissue. A limitation of this study is that we did not directly determine TnI phosphorylation in ischemic and perfused regions of the left ventricular free wall.…”

Cardiac AAV9 Gene Delivery Strategies in Adult Canines: Assessment by Long-term Serial SPECT Imaging of Sodium Iodide Symporter Expression

“…Protein extracts (30 μg per lane) were prepared in sample buffer (12.5% glycerol, 0.1 M Bis-Tris pH7, 1% LDS, 0.5 mmol/l EDTA, and 5 mmol/l tris(2-carboxyethyl)phosphine), resolved by 29 : 1 10% SDS-PAGE and transferred to polyvinylidene fluoride membrane as described earlier. 36 Membranes were blocked in PBS-T including 3% (w/v) BSA for 1 hour at room temperature and incubated overnight at 4 °C with a custom made, affinity purified anti-cNIS rabbit polyclonal antibody. The antibody was prepared by immunizing rabbits by DNA vaccination technology using a sequence encoding amino acids 546-642 of the canine NIS protein (GenBank XP_541946.3), and the collected serum was affinity-purified against the recombinant antigen to obtain the purified polyclonal antibody (Sdix, Newark, DE).…”