Effects Of Oxypurinol On Contractility In Failing Mouse Myocardium

Objective Congestive heart failure (CHF) is an important cause of mortality throughout the world. Oxidant stress has been implicated in heart failure. The formation of oxygen-derived free radicals is enhanced in congestive heart failure. Xanthine oxidase (XO) is likely to contribute to superoxide radical (O^-) generation in CHF. In this study, acute myocardium infarction (AMI)/heart failure rats were used as a CHF model to investigate the effect of long-term oral treatment with oxypurinol, a XO inhibitor, on contractility and Ca²⁺ transients. Calcium cycling and myofilaments to Ca²⁺ sensitivity in heart failure myocardium was also investigated. We also investigated whether such effect is due to suppression of oxidant stress. Methods 35 male SV129 mice, weighted 22.0-27.1g, were divided randomly into 3 groups: sham (n=10), MI (n=13), and MI+ oxypurinol (n=12). Myocardium infarction (MI) mouse model was made by occluded left anterior descending coronary artery. Sham mice were undergone the same operation without artery occlusion. Oxypurinol was administrated orally (1.0 mmol/L) in their drinking water after MI operation. Echocardiography was used to assay the cardiac structure and function of the mice with heart failure MI. Left ventricular (LV) dimension and function parameters (LV dimensions and fractional shortening of LV wall) were measured with echocardiography. Trabecular muscles from 3 groups of hearts were dissected: sham (n=6), MI (n=8), and MI + Oxy (n=7). Force and [Ca²⁺]i were measured simultaneously in fura-2 loaded intact trabeculae from the right ventricle at various extracellular calcium and various physiological stimulative frequency . Trabecular were mounted between a force transducer and a hood and superfused with K-H solution at 22°C. Fura-2 pentapotassium salt, fluoresent tracer of calcium was microinjected into 2 to 4 cells to achieve a fluorescence level that was 3-4 times the auto fluorescence level and allowed to spread throughout the preparation via gap junctions. Fura-2 fluorescence was excited at 340 and 380nm. [Ca²⁺]_i was determined from the following after subtracting autofluorescence: [Ca²⁺]_i= K'_d (R-R_min)/(R_max- R), where R was the observed ratio of fluorescene (340/380), K'_d was the apparent dissociation constant, R_max was the ratio 340/380 nm at saturating [Ca²⁺]_o, and R_min was the ration 340/380 nm at zero [Ca²⁺]_o. Values for K'_d, R_max, R_min were obtained by in vivo calcium calibration. K'_d R_max and R_min were 3.3, 10.33±0.65 and 0.23±0.02 respectively. Steady-state force-[Ca²⁺]_i relations was obtained by tetanizing the trabecular in the presence of ryanodine (1umol/L) at various extracellular Ca²⁺ concentrations. Steady state force -[Ca²⁺]_i relations were fitted with a function of the following form (Hill equation): F=F_max[Ca²⁺]_n/(Ca₅₀ⁿ +[Ca²⁺]_n). where n is the Hill coefficient, F_max is the maximal Ca²⁺ activated force, Caso is the [Ca²⁺]_i required for 50% of maximal activation. XO activity was measured by using the Amplex(?) Red Xanthine/Xanthine Oxidase Assay Kit. Lipid peroxidation was assayed by a colorimetric reaction between MDA, a lipid peroxidation product, and thiobarbituric acid (TBA), which formed TBARS. The data was normalized to the cell protein content. Total protein of myocardium homogenate was measured using Pierce BCA protein assay kit. Myofibrillar proteins oxidation were measured as protein carbonyl groups using the OxyBlot^TM protein oxidation detection kit to measure carbonyl levels in individual proteins within a mixture of proteins (myocardium homogenize) by reaction with dinitrophenylhydrazine (DNPH) followed by western blot immunoassay. Results Significant lack of posterior wall motion in MI-control mice compared with sham group. Improved posterior wall motion in oxypurinol treated mice compared with MI-control group. There was a significant difference between MI mice and SHAM mice (p＜0.05) in left ventricular dimension. There was a slight decrease of the left ventricular dimension in the mice heart treated with oxypurinol. Both fraction of LV shortening (%) (oxy+MI group: 29.55±2.58 vs MI group: 19.88±1.73) and fraction of wall thickness (%)(34.14±3.28 vs 25.88±2.42)were increased significantly in the mice heart treated with oxypurinol (p＜0.05 vs MI mice). In MI group, both twitch force and [Ca²⁺]_i transient were significantly decreased than that in sham group (p＜0.05). Both twitch force and [Ca²⁺]_i transient were significantly increased in oxypurinol treated group compared with MI group (p＜0.05). The twitch tensions were enhanced 98%, 90%, 50% in oxypurinol-treated muscle and the [Ca²⁺]_i transient were increased 99%, 85%, 41% in oxypurinol-treated muscle when [Ca²⁺]_o were 1, 5, 10 mmol/L, respectively. In the heart failure muscle, both twitch force (34.45±3.37mN/mm²) and [Ca²⁺]_i transient (0.54±0.05μM) were significant decreased at higher [Ca²⁺]_o concentration (10 mM), vs. sham group twitch force (70.29±6.72mN/mm²) and [Ca²⁺]_i transient (1.05±0.10μM) (p＜0.05). The systolic force (60.22±6.02mN/mm²) in oxypurinol treated mice increased significantly compared with that of MI mice. At the same time, the systolic [Ca²⁺]_i (0.82+4±0.08μM) in oxypurinol treated mice increased significantly compared with that of MI mice. However, the diastolic force did not raise although there was a slight increase of the diastolic [Ca²⁺]_i. Oxypurinol increased systolic force did not change diastolic force, an excellent character distinguishing it from traditional positive inotropic drugs, which increased diastolic force as well as systolic force and as a result, aggravated the impairment of the myocardium. When stimulation frequency was increased from 0.5 to 5Hz, the nonfailing (sham) myocardium responded with a pronounced increase in the amplitude of calcium (from 0.42±0.04μM to 1.24±0.09μM). The increase in [Ca²⁺]_i was accompanied by an increase in twitch tension amplitude (from 12.19±1.72 mN/mm² at 0.5Hz to 43.39±3.1mN/mm² at 5Hz)and minimal changes in diastolic tension. In the failing myocardium, twitch tension was maximal 16.23±1.62 mN/mm² at 3Hz and then declined to 6.84±0.73 mN/mm² at 5Hz. This decline in force was associated with a parallel decline in the Ca²⁺ transients from 0.51±0.05μM at 3Hz to 0.45±0.05μM at 5Hz. The diastolic force and the diastolic [Ca²⁺]_i transient were also influenced by frequency. At lower frequency (0.5-2 HZ) stimulation, there was no change among 3 groups. But at higher frequency (3-5HZ) stimulation, both the diastolic force, and the diastolic [Ca²⁺]_i were increased slightly but the systolic force decreased significantly in MI muscle. In OXY treated MI muscles, the change of twitch tension and [Ca²⁺]_i with the stimulation frequency increasing was similar to Sham muscles. Oxypurinol improved force-frequency relationship by improving Ca²⁺ cycling in HF and thus recovering Ca²⁺ loading in SR. Steady-state force was significantly enhanced in oxypurinol treated group compared with MI group despite of similar [Ca²⁺]_i concentration (0～2.5 umol/L). The maximum Ca²⁺-force was nearly doubled from (oxy+MI group: 74±4.5mN/mm²) to (MI group: 32±2.5mN/mm²), which indicated that a markedly elevated maximum force generating capacity, underlied the increase in twitch tension treated by oxypurinol. The force middle point [Ca²⁺]_i had no significant change between MI and oxypurinol treated group(from 0.48 to 0.47 umol/L). XO activity was up-regulated in heart failure myocardium following MI (sham 2.92±0.29, MI 5.53±0.41, MI+OXY 2.63±0.27 mU/mg protein), XO activity was decreased in oxypurinol treated group (p＜0.01). TBARS content was increased significantly in MI-control group (0.57±0.05 nmol/mg protein) (p＜0.05). TBARS content was reduced significantly in MI-oxypurinol treated group (0.71±0.06 nmol/mg protein) (p＜0.05). TBARS content was reduced significantly in MI-oxypurinol treated group (0.72±0.05 nmol/mg protein) compared with MI group (p＜0.05). It implied that long term treatment with oxypurinol could inhibit XO activity, reduce lipid peroxidation and as a result lead to restoration of cardiac function. In MI group, oxidation protein level was significantly increased. There was oxidative change in protein structure in the level of actin-modified protein. In oxypurinol treated group, oxidation proteins significantly deceased. Similar results were obtained in sham group. The modified proteins had not been identified. Whether this abnormal oxidation proteins was a marker or result of oxidative stress leading to protein denaturation and enzyme inactivation were unclear. The abnormal calcium cycling and myofilaments to Ca²⁺ sensitivity in heart failure myocardium may be involved in lipid peroxidation and/or oxidation proteins. It revealed that ROS played a pathophysiological role in cardiac tissue damage. Conclusion Progression to later stages of HF was characterized by contractile dysfunction, increased oxidant stress and reduced antioxidant reserve. Antioxidation effect of oxypurinol attenuated XO activity, diminished oxygen-generated free radical formation and thus prevented myocardium from lipid peroxidation and protein oxidation in failing mouse after acute myocardial infarction. Orally administrated oxypurinol could ameliorate abnormal excitation contraction coupling and improve cardiac function in failing heart by altering Ca²⁺ cycling and recovering myofilament calcium responsiveness. It had an ideal positive inotropic effect on mouse heart failure after myocardial infarction. The present study hinted that negative inotropic effect was associated with lipid peroxidation and protein oxidation induced by XO^- generated free radical in failing myocardium. Oxypurinol, a XO inhibitor, was a potential strategy for the treatment of human heart failure. This study was supported by the National Institutes of Health grants R01-HL-44065.