Evaluation of thyroid hormone induced pharmacological preconditioning on cardiomyocyte protection against ischemic-reperfusion injury.

OBJECTIVES: Ischemic preconditioning (IPC) has been demonstrated to make myocardium transiently more resistant to deleterious effect of prolonged ischemia. The opening of the mitochondrial permeability transition pore (mPTP) at the time of myocardial reperfusion is a critical determinant of cell death. L-thyroxine pre-treatment increases the tolerance of the heart to ischemia and produces cardioprotection similar to ischemic precondition. This study has been designed to investigate the mechanism involved in L-thyroxine-induced cardiomyocyte protection against ischemia-reperfusion (I/R) injury in rats.
MATERIALS AND METHODS: L-thyroxine (T(4)) was administered to Wistar rats (n=6) (25 μg/100 g/day s.c.) for two weeks. Hearts from normal and L-thyroxine-treated rats were perfused in Langendorff's mode and subjected to 30 min of ischemia followed by 120 min of reperfusion. Myocardial infarct size was estimated by triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was analyzed in coronary effluent.
RESULTS: IPC and pharmacological preconditioning (PPC) significantly decreased (P<0.05) myocardial infarct size, release of LDH and CK-MB in rat heart. Perfusion of atractyloside, an opener of mPTP, significantly (P<0.05) attenuated the cardioprotective effect of IPC and L-thyroxine-induced pharmacological preconditioning (PPC) in normal rat heart.
CONCLUSION: The cardioprotective effect of L-thyroxine-induced preconditioning may be mediated through inhibition of mPTP opening during reperfusion phase.

Introduction

Ischemic heart disease is a leading cause of morbidity and mortality worldwide.[1] Ischemic preconditioning (IPC) is a well-described adaptive response in which brief exposure to ischemia markedly enhances the ability of the heart to withstand a subsequent ischemic insult.[2] In fact, preconditioning mechanisms could potentially be feasible paradigms for the development of new pharmacologic approaches. Numerous pharmacologic agents such as adenosine, bradykinin, nitric oxide (NO) and α-adrenergic agonist have been demonstrated to elicit pharmacological preconditioning.[3] Recently, the cardioprotective effect of thyroid hormone (TH) has been demonstrated in cells, animals and humans.[4] Acute and long-term TH treatment is known to protect the myocardium from ischemia-reperfusion injury. Moreover, long-term treatment with L-thyroxine has been demonstrated to up regulate protein kinase C-delta (PKC-δ) and produce cardioprotection,[45] activation of PKC is known to inhibit opening of mitochondrial permeability transition pore (mPTP) by opening of potassium ATP channels (KATP).[6] It has been reported that mPTP opens during reperfusion injury due to oxidative stress, Ca++ overload, decreased ATP levels, and increased matrix pH.[78] mPTP opening causes uncoupling of oxidative phosphorylation and decreases mitochondrial ATP level.[9] L-thyroxine-treated hearts seem to display a phenotype of cardioprotection that needs to be characterized further. Therefore, this study has been designed to investigate the possible mechanism involved in L-thyroxine-induced cardiomyocyte protection against ischemic reperfusion (I/R) injury.

Materials and Methods

Wister rats of either sex, weighing 250-300 g were employed. The animal experiments were conducted in accordance with guidelines of US National Institute of Health for care and use of laboratory animals and the study protocol was approved by Institutional Ethics Committee.

Drugs and chemicals

L-thyroxine (T4) (Sigma Aldrich [P] Ltd., Bangalore, India) was dissolved in 99% ethanol by adding a small volume (20 μl) of 25% NaOH and diluted 33 times by adding 0.9% NaCl to obtain a 1mg/ml. Before each injection, a fresh solution was made in 0.9% NaCl to a concentration of 50 μg T4/ml. The dose of L-thyroxine (25 μg /100 g body weight) was selected on the basis of previous study[4] and administered subcutaneously once daily for 14 days. Atractyloside potassium (Sigma Aldrich [P] Ltd., Bangalore, India) was dissolved in minimum quantity of distilled water and added to Kreb's–Henseleit (K-H) solution. All other reagents were of analytical grade and were freshly prepared before use.

Isolated rat heart preparation

Rats were administered heparin (500 IU/L, i.p) 20 min prior to sacrificing the animal by cervical dislocation. Heart was rapidly excised and immediately mounted on Langendorff's apparatus.[1011] Isolated heart was retrogradely perfused at constant pressure of 80 mmHg with Kreb′s-Henseleit (K-H) buffer (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5mM; MgSO4.7H2O 1.2mM; KH2PO4 1.2mM; glucose 11mM), pH 7.4, maintained at 37°C bubbled with 95% O2 and 5% CO2. Flow rate was maintained at 7-9 ml/min. using Hoffman's screw. The heart was enclosed in double wall jacket and the temperature was maintained at 37°C by circulating water. Global ischemia was produced for 30 min by blocking the inflow of K-H solution followed by 120 min. of reperfusion. Coronary effluent was collected before ischemia, immediately, 5 min. and 30 min. after reperfusion for estimation of lactate dehydrogenase (LDH)[12] and creatine kinase (CK-MB).[13]

Measurement of thyroid hormones (T4)

Plasma T4 quantitative measurements were performed with ELISA, using kits obtained from Apollo Diagnostic, lab, Moga, Punjab (No 1100 for total T4), as previously described.[14] T4 levels were expressed as ng/ml of plasma. Absorbance measurements were performed at 450 nm with TecanGenios ELISA reader (Tecan, Austria).

Assessment of myocardial infarct size

Infarct size was measured by macroscopic method using TTC-staining dye and the infracted area reported as the percentage of total ventricular area.[15] Hearts were removed from the Langendorff's apparatus, the auricles and the root of the aorta were excised, and the ventricles were frozen. These were then sliced into uniform sections of 2-3 mm thickness and incubated in 1% triphenyltetrazolium chloride, at 37°C in 0.2M Tris buffer (pH 7.4), for 20 min. TTC was converted to red formazone pigment by reduced nicotinamide adenine dinucleotide (NADH) and dehydrogenase enzyme and, therefore, stained the viable cells deep red, while the infracted cells remained unstained or dull yellow. The ventricular slices were placed between two glass plates and a transparent plastic grid with 100 squares in 1 cm2 was placed above it. The average area of each slice was calculated by counting the number of squares on either side. Similarly numbers of square falling over non-stained dull yellow area were counted. Infarct size was expressed as percentage of average ventricular area of both slides of slice. Whole of ventricle slices were weighed, infracted dull yellow part was dissected out, weighed and infarct size was expressed as a percentage of total ventricular weight.

To determine the extent of myocardial injury, the release of LDH and CK-MB in coronary effluents was measured using commercially available kit (Sigma Aldrich, Bangalore, India). Values were expressed in international units per litre (IU/L).

Estimation of lactate dehydrogenase (LDH)

LDH was estimated in coronary effluent collected immediately and 30 min after reperfusion using 2, 4- DNPH method as described by King, 1959.[12]

Estimation of creatine kinase (CK-MB)

CK-MB was measured in coronary effluent after stabilization and 5 min after reperfusion using modified method of Hughes, 1962.[13]

Experimental protocol

Eight group, each consisted of six rats, were used. In all groups, the isolated rat heart was perfused with K-H solution and stabilized for 10 min and coronary effluent was collected for 1 min, immediately after stabilization (basal), 5 min, and 30 min after reperfusion for LDH and CK estimation.

Group-I: (Sham control): Isolated rat heart was perfused continuously for 200 min without subjecting them to global ischemia and reperfusion.

Group-II: (L-thyroxine (T4) per se): L-thyroxine pre-treated rat heart was perfused continuously for 200 min without subjecting to zero-flow global ischemia and reperfusion.

Group-III: (atractyloside per se): Rat heart was perfused with atractyloside (20 μM) for 5 min after stabilization followed by global reperfusion up to 200 min.

Group-IV: (ischemia-reperfusion control): Isolated rat heart preparation was perfused for 40 min with K-H buffer solution. Then the preparation was subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group-V: (ischemic preconditioning control): Isolated rat heart preparation was subjected to four cycles of IPC. Each cycle comprised of 5 min ischemia followed by 5 min reperfusion with K-H buffer solution. The heart was then subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group-VI: (IPC and atractyloside (20 μM) treated normal heart): Heart obtained from normal rat was reperfused with atractyloside (20 μM) in the last episode of reperfusion of IPC. Then the preparation was subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group-VII: (L-thyroxine-treated and I/R): L-thyroxine pre-treated rat heart preparation was perfused for 40 min with K-H buffer solution. Then the preparation was subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group-VIII: (L-thyroxine pre-treated and atractyloside (20 μM) treated heart): L-thyroxine pre-treated rat heart preparation was perfused for 40 min with K-H buffer solution, rat heart was reperfused with atractyloside (20 μM) before 30 min global ischemia followed by 120 min of reperfusion.

Statistical analysis

Data were expressed as mean ± standard deviation (S.D). The data was analyzed using analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. P<0.05 was considered as statistically significant.

Results

Effect of L-thyroxine (T4) on thyroid hormone level

The total plasma T4 level was significantly (P<0.05) increased in rats treated with L-thyroxine (25 μg/100 g s.c., daily) for two weeks as compared with untreated rats.

Effect of ischemic preconditioning in ischemic–reperfusion injury of rat heart

I/R injury produced a significant increase (P< 0.05) in myocardial infarct size [Figure 1], release of CK-MB and LDH [Table 1] in coronary effluent in the normal rat heart, as compared to sham control group (Group-I). IPC significantly reduced ischemia-reperfusion induced myocardial infarct size and release of CK-MB and LDH in normal rat heart and release of CK-MB and LDH in normal rat hearts.

Figure 1

Effect of L-thyroxine pre-treatment and ischemic preconditioning on myocardial infarct size in ischemic –reperfusion injury in rat heart

Table 1

Effect of ischemic preconditioning on I/R injury induced CK-MB release in normal and L-thyroxine treated rat heart

Effect of atractyloside on cardioprotective effect of ischemic preconditioning

Administration of atractyloside 5 min before four cycle of 5 min ischemia and 5 min reperfusion (IPC) significantly attenuated (P<0.05) the cardioprotective effect of IPC assessed in terms of myocardial infarct size [Figure 1], CK-MB and LDH [Table 1].

Effect of pre-treatment with thyroxine on ischemic-reperfusion injury of rat hearts

PPC significantly (P<0.05) reduced the I/R-induced myocardial infarct size [Figure 1], and release of CK-MB and LDH [Table 1] in coronary effluent as compared with untreated rat heart.

Effect of atractyloside on cardioprotective effect of PPC with L-thyroxine

Atractyloside (20 μmol/L) significantly (P<0.05) attenuated the pharmacological preconditioning induced cardioprotection by L-thyroxine (25 μg/100 g s.c), measured in terms of myocardial infarct size [Figure 1], release of CK-MB and LDH [Table 1] in coronary effluent of isolated rat heart. Atractyloside (20 μmol/L) and L-thyroxine(25 μg/100 g s.c) administered separately and in combination did not affect coronary flow rate (data not shown).

Discussion

Thyroid hormone (TH) can regulate important cardioprotective signalling and may increase tolerance of the myocardium to I/R injury. Post-ischemic recovery was found to be increased after long-term L-thyroxine pre-treatment and ischemic preconditioning, as compared to untreated hearts. This study has demonstrated that L-thyroxine-induced pharmacological preconditioning and ischemic preconditioning can display a similar pattern of cardioprotection against ischemia-reperfusion injury. The transient occlusion of circumflex artery has been reported to produce protection of myocardial region supplied by left anterior descending coronary artery.[1617] Similarly, in the present study, ischemic preconditioning and L-thyroxine-induced pharmacological preconditioning significantly attenuated ischemia and reperfusion-induced increase in myocardial infarct size and release of CK-MB and LDH. This observation is consistent with our previous reports.[318] Langendorff's preparation and working heart preparation are hemodynamically comparable to investigate the effect of pharmacological agents on ischemia and reperfusion induced myocardial injury.[1119] Moreover, Langendorff's preparation permits the use of pharmacological interventions without any interference due to changes in systemic circulation.[20] Therefore, the isolated rat heart preparation perfused retrogradely on Langendorff's apparatus was employed in the present study.

The cardioprotective effect of TH has been demonstrated in cells, animals and humans.[42122] Acute and long-term TH treatment has been shown to protect the myocardium from ischemia–reperfusion injury. In addition, thyroid hormone pre-treatment was also shown to confer protection against lethal ischemia, in a pattern, similar to that occurred in ischemic preconditioning.[4] However, the mechanism involved in L-thyroxine-induced cardiomyocyte protection is not still explored. Thyroid hormone critically regulates cardiac performance, since several genes encoding important structural and regulatory proteins in the myocardium, including myosinisoform expression, calcium cycling proteins, and protein kinases (such as PKC-ε and PKC-δ known to phosphorylate cardiaccontractile proteins) are thyroid hormone responsive.[422] Thus, thyroid analogues are suggested as potential therapeutic agents for treating heart failure. PKC is an intracellular molecule that has been recognized to have an important role in ischemic and pharmacologic preconditioning.[56] Specifically, the ε and δ isoforms of PKC have been identified to be involved in ischemic preconditioning mediated cardioprotection.[23] PKC alterations are found to occur after long-term L-thyroxine administration. In fact, in an earlier study, it has been demonstrated that the basal expression of PKCe was decreased in L-thyroxine-treated hearts.[24] Recently, it has been demonstrated that transfected neonatal rat cardiac myocytes that over-express PKC-δ are more resistant to ischemic injury and this study suggested that activation or over-expression of PKC-δ mimic ischemic preconditioning. Furthermore, PKC can phosphorylate important cardioprotective molecules such as heat shock protein HSP27.[25] It seems likely that PKC-δ and PKCε may serve a key role in the response of the hyperthyroid and hypothyroid myocardium to ischemia–reperfusion injury. Thus administration of L-thyroxine up-regulates PKCε/δ expression that consequently preconditions the heart. PKC has been suggested to produce cardioprotection by opening of mitochondrial ATP-sensitive K+ channel (mito K).[6] PKC-ε as well as PKC-δ has been demonstrated to mimic preconditioning due to opening of mito K channel.[5] Further, opening of mito K channel depolarizes inner mitochondrial membrane and reduce calcium entry into the mitochondria resulting in inhibition of opening of mPTP[9] and a consequent decrease in the release of cytochrome C and reduction of apoptotic cell death. Therefore, it was speculated that L-thyroxine treatment modulates various cellular events at molecular levels that converge and inhibits the opening of mPTP and produced cardiomyocyte protection. The attenuation of L-thyroxine-induced pharmacological preconditioning by atractyloside, an mPTP opener, observed in the present study confirms this hypothesis. Our result is fully consistent with previous report by Pantos et al., that observed ischemic preconditioning like cardioprotection with thyroxine treatment. Moreover, thyroid-induced preconditioning has been also demonstrated in clinical practice in patients subjected to CABG and cardiac failure.[22]

Conclusion

These results suggest that cardioprotective effect of L-thyroxine-induced preconditioning may be mediated through inhibition of mPTP opening during reperfusion phase. L-thyroxine might prove as a suitable pharmacologic agent that could maintain the heart in preconditioned state.