Role of proteases in the pathophysiology of cardiac disease

Cardiovascular disease is a major cause of death and thus a great deal of effort has been made in salvaging the diseased myocardium. Although various factors have been identified as possible causes of different cardiac diseases such as heart failure and ischemic heart disease, there is a real need to elucidate their role for the better understanding of the cardiac disease pathology and formulation of strategies for developing newer therapeutic interventions. In view of the intimate involvement of different types of proteases in maintaining cellular structure, the role of proteases in various cardiac diseases has become the focus of recent research. Proteases are present in the cytosol as well as are localized in a number of subcellular organelles in the cell. These are known to use extracellular matrix, cytoskeletal, sarcolemmal, sarcoplasmic reticular, mitochondrial and myofibrillar proteins as substrates. Work from different laboratories using a wide variety of techniques has shown that the activation of proteases causes alterations of a number of specific proteins leading to subcellular remodeling and cardiac dysfunction. Inhibition of protease action by different drugs and agents, therefore, has a clinical relevance and is expected to form a part of new treatment paradigm for improving heart function. This review examines the biochemistry and localization of some of the proteases in the cardiac tissue in addition to identification of the sites of action of some protease inhibitors. (Mol Cell Biochem 263: 241–256, 2004)     

Decreased expression of cardiac sarcoplasmic reticulum Ca2+-pump ATPase in congestive heart failure due to myocardial infarction

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca²⁺-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca²⁺-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca²⁺-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.     

Diabetes-like action of intermittent fasting on sarcoplasmic reticulum Ca2+-pump ATPase and myosin isoenzymes can be prevented by sucrose

Experimental diabetes results in a reduction of the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase activity and a redirection of myosin isoenzymes from V1 to V3. Similar, but less pronounced, changes were induced by subjecting rats to intermittent fasting for 6 weeks. Low amounts of sucrose (0.8%) in the drinking water prevented the subcellular changes in fasted rats; however, sucrose neither affected the levels of plasma thyroid hormones nor normalized the reduced body weight. Plasma glucose was lowered without any changes in plasma insulin in the fasted rats receiving sucrose; this suggested an enhanced peripheral glucose utilization. Thus, the signals in the diabetic heart leading to changes in SR and myosin can be mimicked by intermittent fasting and seem to be linked to a shift in fuel utilization by the myocytes.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Mechanisms of cardiodepression by an Na+-H+ exchange inhibitor methyl-N-isobutyl amiloride (MIA) on the heart: lack of beneficial effects in ischemia-reperfusion injury

Although Na+-H+ exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular Ca2+ mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HCO3- -free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HCO3-containing buffer. MIA increased the basal concentration of intracellular Ca2+ ([Ca2+]i) and augmented the KCl-mediated increase in [Ca2+]i. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils Ca2+-stimulated ATPase activity. MIA also increased Ca2+ uptake by mitochondria without having any direct effect on sarcoplasmic reticulum Ca2+ uptake. In addition, MIA did not protect the hearts subjected to mild Ca2+ paradox as well as ischemia-reperfusion-mediated injury. These results suggest that the increase in [Ca2+]i in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial Ca2+ overloading as well as indirect depression of myofibrillar Ca2+ ATPase activity. Thus the accumulation of [H+]i as well as occurrence of intracellular and mitochondrial Ca2+ overload may explain the lack of beneficial effects of MIA in preventing the ischemia-reperfusion-induced myocardial injury.     

Suppression of high lipid diet induced by atherosclerosis sarpogrelate

Sarpogrelate (SP), a serotonin (5‐HT2A) receptor antagonist, is used as an anti‐platelet agent for the treatment of some vascular diseases. SP has been reported to inhibit 5‐HT induced coronary artery spasm, increase in intracellular calcium and smooth muscle cells proliferation. This study was undertaken to test that SP suppresses the development of atherosclerosis due to high cholesterol diet (HCD) by decreasing blood viscosity and oxidative stress. For this purpose, 29 rabbits were divided into four groups: control group (normal diet); normal diet group with SP at the dose of 5 mg/kg/day; HCD group fed 1% cholesterol; and HCD group with SP at the dose of 5 mg/kg/day. After 90 days of the experiment, blood samples were collected and the animals were killed; the thoracic aorta was stained by the Oil Red O staining method. The results indicate that plasma levels of cholesterol, triglycerides and malondialdehyde were increased in rabbits fed HCD. Plasma viscosity and whole blood viscosity were also higher in the HCD group than that in normal diet group. Treatment with SP prevented these alterations induced by HCD whereas this agent had no significant effect in rabbits fed normal diet. Morphological examination of the aorta revealed that SP treatment prevented the formation of foam cells and atherosclerotic plaque. It is suggested that the beneficial effects of SP in atherosclerosis may be due to actions on blood viscosity, lipid levels and oxidative stress.     

Reversal of subcellular remodelling by losartan in heart failure due to myocardial infarction

This study tested the reversal of subcellular remodelling in heart failure due to myocardial infarction (MI) upon treatment with losartan, an angiotensin II receptor antagonist. Twelve weeks after inducing MI, rats were treated with or without losartan (20 mg/kg; daily) for 8 weeks and assessed for cardiac function, cardiac remodelling, subcellular alterations and plasma catecholamines. Cardiac hypertrophy and lung congestion in 20 weeks MI‐induced heart failure were associated with increases in plasma catecholamine levels. Haemodynamic examination revealed depressed cardiac function, whereas echocardiographic analysis showed impaired cardiac performance and marked increases in left ventricle wall thickness and chamber dilatation at 20 weeks of inducing MI. These changes in cardiac function, cardiac remodelling and plasma dopamine levels in heart failure were partially or fully reversed by losartan. Sarcoplasmic reticular (SR) Ca²⁺‐pump activity and protein expression, protein and gene expression for phospholamban, as well as myofibrillar (MF) Ca²⁺‐stimulated ATPase activity and α‐myosin heavy chain mRNA levels were depressed, whereas β‐myosin heavy chain expression was increased in failing hearts; these alterations were partially reversed by losartan. Although SR Ca²⁺‐release activity and mRNA levels for SR Ca²⁺‐pump were decreased in failing heart, these changes were not reversed upon losartan treatment; no changes in mRNA levels for SR Ca²⁺‐release channels were observed in untreated or treated heart failure. These results suggest that the partial improvement of cardiac performance in heart failure due to MI by losartan treatment is associated with partial reversal of cardiac remodelling as well as partial recovery of SR and MF functions.     

Differences in ischemia-reperfusion-induced endothelial changes in hearts perfused at constant flow and constant pressure

Isolated hearts subjected to ischemia-reperfusion (I/R) exhibit depressed cardiac performance and alterations in subcellular function. Since hearts perfused at constant flow (CF) and constant pressure (CP) show differences in their contractile response to I/R, this study was undertaken to examine mechanisms responsible for these I/R-induced alterations in CF-perfused and CP-perfused hearts. Rat hearts, perfused at CF (10 ml/min) or CP (80 mmHg), were subjected to I/R (30 min global ischemia followed by 60 min reperfusion), and changes in cardiac function as well as sarcolemmal (SL) Na(+)-K(+)-ATPase activity, sarcoplasmic reticulum (SR) Ca(2+) uptake, and endothelial function were monitored. The I/R-induced depressions in cardiac function, SL Na(+)-K(+)-ATPase, and SR Ca(2+)-uptake activities were greater in hearts perfused at CF than in hearts perfused at CP. In hearts perfused at CF, I/R-induced increase in calpain activity and decrease in nitric oxide (NO) synthase (endothelial NO synthase) protein content in the heart as well as decrease in NO concentration of the perfusate were greater than in hearts perfused at CP. These changes in contractile activity and biochemical parameters due to I/R in hearts perfused at CF were attenuated by treatment with l-arginine, a substrate for NO synthase, while those in hearts perfused at CP were augmented by treatment with N(G)-nitro-l-arginine methyl ester, an inhibitor of NO synthase. The results indicate that the I/R-induced differences in contractile responses and alterations in subcellular organelles between hearts perfused at CF and CP may partly be attributed to greater endothelial dysfunction in CF-perfused hearts than that in CP-perfused hearts.