Abstract 39: Organic Nitrates Improve Calcium Handling and Myofilament Sensitivity Impaired by Nitroso-Redox Disequilibrium

Oxidative stress is a key factor in the dysfunctional calcium (Ca 2+ ) handling and contractile performance in heart disease. Organic nitrates are used for the treatment of cardiovascular afflictions, however endogenous nitric oxide (NO) has a broad spectrum of actions and the mechanisms by which it regulates Ca 2+ cycling and contractility is poorly understood. Neuronal NO synthase (nNOS) deficiency induces a nitroso-redox disequilibrium characterized by oxidative stress and altered contractility and calcium handling. We hypothesize that treatment of nNOS −/− mice with organic nitrates such as nitroglycerin (TNG) or isosorbide dinitrate (ISDN) reduces cytosolic calcium levels and restores myofilament responsiveness to calcium with no changes in contractility. Cardiomyocytes (CMs) were isolated from nNOS −/− (N=7) and wild type (WT) mice (N=3). Cells were loaded with fura-2 and then electrically evoked intracellular Ca 2+ and sarcomere length were simultaneously measured in an IonOptix system. It has been shown that L-type Ca 2+ current is exacerbated in nNOS −/− cardiomyocytes. In consequence, at 1 Hz pacing rate, Ca 2+ peak and transient amplitude were increased in nNOS −/− (Peak Ca 2+ : 571 ± 38 nM) compared to WT (401 ± 32 nM; p = 0.012) cardiomyocytes, which was reduced toward normal by 10 nmol/L TNG (453 ± 55 nM; p = 0.11) as well as 10 nmol/L ISDN (394 ± 65; p = 0.035). Phospholamban phosphorylation has been shown to be reduced in nNOS −/− CMs, possibly due to the enhanced activity of protein phosphatases induced by oxidative stress. Thus, Ca 2+ decay and sarcomere relaxation were slower in nNOS −/− compared to WT CMs ( p < 0.0001). Treatment with TNG ( p < 0.0001 vs. NOS1 −/− ) or ISDN ( p < 0.0001 vs. NOS1 −/− ) accelerated Ca 2+ decay and relaxation. Ca 2+ sensitivity was impaired in nNOS −/− ( p < 0.021 vs. WT). Although organic nitrates either reduce or do not affect myofilament sensitivity in normal myocytes, TNG and ISDN increased their responsiveness to Ca 2+ in nNOS −/− . In conclusion, restoration of NO availability and subsequent attenuation of the nitroso-redox imbalance, improved excitation-contraction coupling in nNOS −/− CMs, decreased intracellular Ca 2+ which is counteracted by the improved myofilament sensitivity resulting in no net change in contractility.

Sarcomere relaxation in hamster diaphragm muscle

We characterized instantaneous sarcomere relaxation over the load continuum in isolated hamster diaphragm muscles by means of laser diffraction. In afterloaded twitches, sarcomere relaxation displayed two consecutive phases. The bulk of sarcomere lengthening occurred during the first phase and corresponded in time to muscle lengthening. The second phase of sarcomere relaxation was slower and corresponded in time to tension decay. At initial muscle length, the peak velocity of sarcomere lengthening (SVL) was linearly related to both the maximum extent of sarcomere shortening (delta SL) and sarcomere length at peak shortening (SLmin; each P < 0.01). Varying preload modified the SVL vs. SLmin relationship but not the SVL vs. delta SL relationship. At a given preload, muscle tension decay began at a similar sarcomere length, regardless of the afterload level. In conclusion, our results support the role played by sarcomere length in regulating the diaphragm muscle-lengthening rate but not the rate of tension decline.

Relaxation abnormalities in single cardiac myocytes from renovascular hypertensive rats

In myocardial hypertrophy secondary to renovascular hypertension, the rate of intracellular Ca2+ concentration decline during relaxation in paced left ventricular (LV) myocytes isolated from hypertensive (Hyp) rats is much slower compared with that from normotensive (Sham) rats. By use of a novel liquid-crystal television-based optical-digital processor capable of performing on-line real-time Fourier transformation and the striated pattern (similar to 1-dimensional diffraction grating) of cardiac muscle cells, sarcomere shortening and relaxation velocities were measured in single Hyp and Sham myocytes 18 h after isolation. There were no differences in resting sarcomere length, percent of maximal shortening, time to peak shortening, and average sarcomere shortening velocity between Sham and Hyp cardiac cells. In contrast, average sarcomere relaxation velocity and half-relaxation time were significantly prolonged in Hyp myocytes. Contractile differences between Sham and Hyp myocytes detected by the optical-digital processor are confirmed by an independent method of video tracking of whole cell length changes during excitation-contraction. Despite the fact that freshly isolated myocytes contract more rigorously than 18-h-old myocytes, the relaxation abnormality was still observed in freshly isolated Hyp myocytes, suggesting impaired relaxation is an intrinsic property of Hyp myocytes rather than changes brought about by short-term culture. We postulate that reduced sarcomere relaxation velocity is a direct consequence of impaired Ca2+ sequestration-extrusion during relaxation in Hyp myocytes and may be responsible for diastolic dysfunction in hypertensive hypertrophic myocardium at the cellular level.