Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.     

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca²⁺ _i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca²⁺ _i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca²⁺ _i levels of the heart. The fluorescence ratio technique was applied to estimate Ca²⁺ _i. The disappearance of mechanical activity of the heart preceded that of the Ca²⁺ _i transient in the first 2 min of ischemia. The slope of upstroke of the Ca²⁺ _i transient, reflecting Ca²⁺ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca²⁺ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca²⁺ _i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca²⁺ _i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca²⁺ _i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca²⁺ _i handling and sustained depression of mechanical performance.     

Ischemic-reperfused isolated working mouse hearts: membrane damage and type IIA phospholipase A2

For the murine heart the relationships between ischemia-reperfusion-induced loss of cardiac function, enzyme release, high-energy phosphate (HEP), and membrane phospholipid metabolism are ill-defined. Accordingly, isolated ejecting murine hearts were subjected to varying periods of ischemia, whether or not followed by reperfusion. On reperfusion, hemodynamic function was almost completely restored after 10 min of ischemia [83 +/- 14% recovery of cardiac output (CO)], but was severely depressed after 15 and 20 min of ischemia (40 +/- 24 and 31 +/- 24% recovery of CO, respectively). Reperfusion was associated with partial recovery of HEP stores and enhanced degradation of phospholipids as indicated by the accumulation of fatty acids (FA). Myocardial FA content and enzyme release during reperfusion were correlated (r = 0.70), suggesting that membrane phospholipid degradation and cellular damage are closely related phenomena. To investigate the role of type IIA secretory phospholipase A2 (sPLA2) in this process, hearts from wild-type and sPLA2-deficient mice were subjected to ischemia-reperfusion. Postischemic functional recovery, ATP depletion, enzyme release, and FA accumulation were not significantly different between wild-type and sPLA2- deficient hearts. These findings argue against a prominent role of type IIA sPLA2 in the development of irreversible cell damage in the ischemic-reperfused murine myocardium.     

Heat stress pretreatment mitigates postischemic arachidonic acid accumulation in rat heart

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage.The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min.Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p < 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.     

Assessment of fatty acids in cardiac tissue as 9-anthryldiazomethane esters by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the rapid assessment of fatty acids in cardiac tissue is described. A level of 50.4 ± 14.9 nmol fatty acids per g wet weight of rat myocardial tissue could be monitored. The content of the individual fatty acids C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_18:2 and C_20:4 amounted to 1.9, 13.5, 0.6, 14.4, 6.1, 6.5 and 7.2 nmol/g wet weight, respectively. A comparison of this method with a well established gas chromatographic technique yielded good agreement. In contrast with time-consuming gas chromatographic techniques, there is no need to isolate (unesterified) fatty acids from the other lipid classes with column chromatography or thin-layer chromatography, because the derivatizing reagent 9-anthryldiazomethane reacts highly specifically with fatty acids.     

Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion

We investigated whether the cardioprotection induced by heat stress (HS) pretreatment is associated with mitigation of phospholipid degradation during the ischemic and/or postischemic period. The hearts, isolated from control rats and from heat-pretreated rats (42 degrees C for 15 min) either 30 min (HS0.5-h) or 24 h (HS24-h) earlier, were subjected to 45 min of no-flow ischemia, followed by 45 min of reperfusion. Unesterified arachidonic acid (AA) accumulation was taken as a measure for phospholipid degradation. Significantly improved postischemic ventricular functional recovery was only found in the HS24-h group. During ischemia, AA accumulated comparably in control and both HS groups. During reperfusion in control and HS0.5-h hearts, AA further accumulated (control hearts from 82 +/- 33 to 109 +/- 51 nmol/g dry wt, not significant; HS-0.5h hearts from 52 +/- 22 to 120 +/- 53 nmol/g dry wt; P < 0.05). In contrast, AA was lower at the end of the reperfusion phase in HS24-h hearts than at the end of the preceding ischemic period (74 +/- 18 vs. 46 +/- 23 nmol/g dry wt; P < 0.05). Thus accelerated reperfusion-induced degradation of phospholipids in control hearts is completely absent in HS24-h hearts. Furthermore, the lack of functional improvement in HS0.5-h hearts is also associated with a lack of beneficial effect on lipid homeostasis. Therefore, it is proposed that enhanced membrane stability during reperfusion is a key mediator in the heat-induced cardioprotection.     

Concomitant accumulation of intracellular free calcium and arachidonic acid in the ischemic-reperfused rat heart

This study was designed to elucidate the relationship between enhanced cytoplasmic calcium levels (Ca²⁺ _i) and membrane phospholipid degradation, a key step in the loss of cellular integrity during cardiac ischemia/reperfusion-induced damage. Isolated rat hearts were subjected to 15 min ischemia followed by 30 min reperfusion. Ca²⁺ _i was estimated by the Indo-1 fluorescence ratio technique. Degradation of membrane phospholipids as indicated by the increase of tissue arachidonic acid content was assessed in tissue samples taken from the myocardium at various points of the ischemia/reperfusion period. The hemodynamic parameters showed almost complete recovery during reperfusion. Fluorescence ratio increased significantly during ischemia, but showed a considerable heart-to-heart variation during reperfusion. Based upon the type of change of fluorescence ratio during reperfusion, the hearts were allotted to two separate subgroups. Normalization of fluorescence ratio was associated with low post-ischemic arachidonic acid levels. In contrast, elevated fluorescence ratio coincided with enhanced arachidonic acid levels. This observation is suggestive for a relationship between the Ca²⁺-related fluorescence ratio and arachidonic acid accumulation probably due to a calcium-mediated stimulation of phospholipase A₂.     

Protein acylation in normoxic and ischemic/reperfused cardiac tissue.

In addition to a prominent role in tissue energy conversion, fatty acids are involved in signal transduction and modulation of cellular protein localization and function. The latter is accomplished by acylation of specific cellular proteins. In the present study the amount of fatty acyl moieties covalently bound to cardiac proteins and the effect of myocardial ischemia and reperfusion on the degree and relative fatty acyl composition of cardiac proteins have been investigated in isolated rat hearts. In the normoxic heart about 0.32% of the cellular fatty acyl pool is covalently bound to proteins. Approximately 90% of these fatty acyl chains are thio-esterified, whereas a relatively minor part is attached to cardiac proteins through amide linkage. Thio-esterified fatty acyl chains are derived from palmitic, stearic, oleic, linoleic, arachidonic and docosahexaenoic acid. In contrast, amide linked protein acylation shows a preference for myristic acyl chains. Acute ischemia and reperfusion inflicted upon the isolated rat heart did enhance significantly the content of (unesterified) fatty acids, but did neither affect the degree of protein acylation nor the relative fatty acyl composition of acylated proteins in cardiac tissue.