Insulin-induced myocardial protection in isolated ischemic rat hearts requires p38 MAPK phosphorylation of Hsp27

Six hours after insulin treatment, hearts express heat shock protein 70 (Hsp70) and have improved contractile function after ischemia-reperfusion injury. In this study we examined hearts 1 h after insulin treatment for contractile function and for expression of Hsp70 and Hsp27. Adult, male Sprague-Dawley rats were assigned to groups: 1) sham, 2) control, 3) insulin injected (200 microU/g body wt), 4) heat shock treated (core body temperature, 42 degrees C for 15 min), and 5) heat shock and insulin treated. At 1 h after these treatments, hearts were isolated, equilibrated to Langendorff perfusion for 30 min, and then subjected for 30 min no-flow global ischemia (37 degrees C) followed by 2 h of reperfusion. Insulin-treated hearts had significantly increased contractile function compared with control hearts. At 1 h after insulin treatment, a minimal change in Hsp70 and Hsp27 content were detected. By 3 h after insulin treatment, a significant increase in Hsp70, but not Hsp27, was detected by Western blot analysis. By immunofluorescence, minimal Hsp70 was detected in insulin-treated hearts, whereas Hsp27 was detected in all hearts, indicative of its constitutive expression. Phosphospecific isoforms of Hsp27 were detected in insulin-, heat shock-, and heat shock and insulin-treated hearts. After ischemia and reperfusion, the insulin-treated hearts had significantly elevated levels of phosphorylated Hsp27. Inhibition of p38 MAPK with SB-203580 blocked the insulin-induced phosphorylation of Hsp27 and the improved functional recovery. In conclusion, insulin induces an apparent rapid phosphorylation of Hsp27 that is associated with improved functional recovery after ischemia-reperfusion injury.     

Myocardial accumulation and localization of the inducible 70-kDa heat shock protein, Hsp70, following exercise

Exercise increases the 70-kDa heat shock protein (Hsp70) in the myocardium, and this exercise-induced increase is associated with significantly improved cardiac recovery following insult. However, while heat shock has been shown to elevate Hsp70 primarily in the cardiac vasculature of the myocardium, the localization following exercise is unknown. Male Sprague-Dawley rats performed continuous treadmill running at 30 m/min for 60 min (2% incline) on either 1 or 5 consecutive days. At 30 min and 24 h following exercise, hearts were extirpated, and the left ventricle was isolated, OCT-cork mounted, and sectioned for immunofluorescent analysis. Whereas immunofluorescent analysis revealed little to no Hsp70 in control hearts and 30 min postexercise, the accumulation of Hsp70 24 h after a single exercise bout or 5 days of training was predominantly located in large blood vessels and, in particular, colocalized with a marker of smooth muscle. Furthermore, higher core temperatures attained during exercise led to more abundant accumulation in smaller vessels and the endothelium. It is concluded that the accumulation of myocardial Hsp70 following acute exercise predominantly occurs in a cell type-specific manner, such that changes in the cardiac vasculature account for much of the increase. This accumulation appears first in the smooth muscle of larger vessels and then increases in smaller vessels and the endothelium, as core temperature attained during exercise increases. This finding supports the observations after heat shock and further suggests that the vasculature is a primary target in exercise-induced cardioprotection.     

The myocardial heat shock response following sodium salicylate treatment

In cultured cells, salicylate has been shown to potentiate the induction of Hsp72 so that a mild heat stress (40 degrees C) in the presence of salicylate induces an Hsp72 response that is similar to a severe heat stress (42 degrees C). To determine whether salicylate can potentiate the myocardial Hsp70 response in vivo and confer protection from an ischemic stress, male Sprague-Dawley rats (250-300 g) were placed into 5 groups: (1) control, (2) salicylate only (400 mg/kg), (3) mild heat stress (40 degrees C for 15 minutes), (4) mild heat stress plus salicylate, and (5) severe heat stress (42 degrees C for 15 minutes). Twenty-four hours following salicylate treatment and/or heat stress, animals were anesthetized, their hearts rapidly isolated, and hemodynamic function evaluated using the Langendorff technique. Hsp72 content was subsequently assessed by Western blotting. Although salicylate in combination with a mild heat stress induced heat shock factor activation, only the hearts from severely heat-stressed animals (42 degrees C) demonstrated a significantly elevated myocardial Hsp72 content and a significantly enhanced postischemic recovery of left ventricular developed pressure and rates of contraction and relaxation. These results support the role for Hsp72 as a protective protein and suggest that neither salicylate treatment alone nor salicylate in combination with a mild heat stress potentiates the myocardial Hsp72 response.     

Hsp70 does not mediate the hyperthermia-associated hyporesponsiveness to angiotensin in isolated rat portal veins

In rat portal veins (RPV) isolated from septic rats, we previously showed that the contractile response to angiotensin II (AT(II)) was significantly decreased and that the vascular failure was correlated with the severity of the disease. We hypothesized that hyperthermia might be one of the factors responsible for the vascular failure. Moreover, hyperthermia should concomitantly increase heat shock proteins (Hsps) expression. We then compared the vascular contractility and the heat shock protein 70 (Hsp70) expression in RPV incubated at 37 degrees C and 39.5 degrees C and sought for a relationship between both events. In our experimental model, hyperthermia increased the Hsp70 expression and decreased the contractile response to AT(II). Incorporation of the Hsp70 antisense oligonucleotide in RPV blocked the increase in Hsp70 expression but had no consequence on the contractile response to AT(II). In conclusion, hyperthermia increases Hsp70 expression but does not mediate the decreased response to AT(II). Hsp70 overexpression has no effect on the actin-myosin interaction in vascular smooth muscle.     

Schisandrin B protects myocardial ischemia-reperfusion injury partly by inducing Hsp25 and Hsp70 expression in rats

Schisandrin B (Sch B) is a hepato- and cardioprotective ingredient isolated from the fruit of Schisandra chinensis, a traditional Chinese herb clinically used to treat viral and chemical hepatitis. In order to investigate whether the induction of heat shock protein (Hsp)25 and Hsp70 expression plays a role in the cardioprotection afforded by Sch B pre-treatment against ischemia-reperfusion (I-R) injury, the time-course of myocardial Hsp25 and Hsp70 expression was examined in Sch B-pre-treated rats. Sch B pre-treatment (1.2 mmol/kg) produced time-dependent increases in Hsp25 and Hsp70 expression in rat hearts, with the maximum enhancement observable at 48 and 72 h post-dosing, respectively. Buthionine sulfoximine/phorone treatment, while abolishing the beneficial effect of Sch B on mitochondrial glutathione redox status, did not completely abrogate the cardioprotection against I-R injury. Heat shock treatment could increase myocardial Hsp25 and Hsp70 expression and protect against I-R injury under the present experimental conditions. The results indicate that the induction of Hsp25 and Hsp70 expression contributes at least partly to the cardioprotection afforded by Sch B pre-treatment against I-R injury.     

Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers

The objective of this study was to investigate the kinetics of Hsp60, Hsp70, Hsp90 protein, and messenger RNA (mRNA) expression levels and to correlate these heat shock protein (Hsp) levels with tissue damage resulting from exposure to high temperatures for varying amounts of time. One hundred broilers were heat-stressed for 0, 2, 3, 5, and 10 h, respectively, by rapidly increasing the ambient temperature from 22 +/- 1 degrees C to 37 +/- 1 degrees C. Obvious elevations of plasma creatine kinase indicate damage to myocardial cells after heat stress. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P < 0.01) at 2 h of heat stress but then decreased quickly after 3 h, while the level of Hsp60 protein in the heart increased (P < 0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicate that the elevation of the three Hsps, especially Hsp60 in heart, may be important markers at the beginning of heat stress and act as protective proteins in adverse environments. The reduction of Hsp signals in the cytoplasm of myocardial cells implies that myocardial cell lesions may have an adverse impact on the function of Hsps during heat stress. Meanwhile, the localization of Hsp70 in blood vessels of broiler hearts suggests another possible mechanism for protection of the heart after heat exposure.     

Exercise training improves myocardial tolerance to ischemia in male but not in female rats

Acute exercise increases myocardial tolerance to ischemia-reperfusion (I-R) injury in male but not in female rat hearts, possibly due to a decreased heat shock protein 70 (Hsp70) response in the female hearts. This study examined whether repetitive exercise training would increase Hsp70 and myocardial tolerance to I-R injury in female rat hearts. Adaptations in myocardial manganese superoxide dismutase (MnSOD) and endothelial nitric oxide synthase (eNOS) were also assessed. Ten-week old male (M) and female (F) Sprague-Dawley rats (n = 40 total) exercise-trained for 14 wk; the last 8 wk consisted of running 1 h at 30 m/min (2% incline), 5 days/wk. Following training, left ventricle mechanical function (LVMF) was monitored for 30 min of reperfusion following 30 min of global ischemia (Langendorff procedure). Myocardial Hsp70 content was not different in M and F control groups, while increases were observed in both trained groups (M greater than F; P < 0.05). Although MnSOD content did not differ between groups, endothelial nitric oxide synthase (eNOS) levels were decreased in F, with no change in M, following training (P < 0.05). Hearts from control F demonstrated a greater recuperation of all indices of LVMF following I-R compared with control M hearts (P < 0.05). Hearts of trained M exhibited improved recovery of LVMF (left ventricular diastolic pressure, left ventricular end-diastolic pressure, +dP/dt, -dP/dt) during reperfusion compared with control M hearts (P < 0.05). In contrast, hearts of trained F did not show any change in recovery from I-R. Hence, exercise training is more beneficial to M than F in improving myocardial function following I-R injury.     

Induced hsp70 is in small, cytoplasmic complexes in a cell culture model of renal ischemia: a comparative study with heat shock

A number of clinical conditions are known to result in the induction of heat shock proteins, but detailed studies on stress response have focused mostly on heat shock as a model. We have analyzed the induction and intracellular distribution of heat shock proteins in a reversible adenosine triphosphate (ATP) depletion model of renal ischemia. Two Hsp70 homologues, Hsp70 in the cytoplasm and BiP in the endoplasmic reticulum (ER) lumen, were found significantly induced during the recovery phase of ATP depletion. Other members of the heat shock protein family, such as Hsp90, constitutive Hsc70, and a related protein Hop60, were not induced. The induction of stress proteins on ATP depletion differed from that after heat shock in the kinds of proteins elaborated, their induction kinetics, and their intracellular distributions. Biochemical fractionation and indirect immunofluorescence experiments indicated that Hsp70 was predominantly cytoplasmic in the recovery phase of ischemia-like stress. Velocity sedimentation on sucrose gradients showed that induced Hsp70 sedimented as small, soluble complexes, ranging in size from 4S20,w to 8S20,w. The results suggest a role for induced Hsp70 that may be different from one of protecting aggregated proteins as under heat shock and emphasize the need for their characterization in other clinical conditions that result in stress response.     

Ischemic preconditioning-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contraction-induced myocardial injury

Dystrophin is an integral membrane protein involved in the stabilization of the sarcolemmal membrane in cardiac muscle. We hypothesized that the loss of membrane dystrophin during ischemia and reperfusion is responsible for contractile force-induced myocardial injury and that cardioprotection afforded by ischemic preconditioning (IPC) is related to the preservation of membrane dystrophin. Isolated and perfused rat hearts were subjected to 30 min of global ischemia, followed by reperfusion with or without the contractile blocker 2,3-butanedione monoxime (BDM). IPC was introduced by three cycles of 5-min ischemia and 5-min reperfusion before the global ischemia. Dystrophin was distributed exclusively in the membrane of myocytes in the normally perfused heart but was redistributed to the myofibril fraction after 30 min of ischemia and was lost from both of these compartments during reperfusion in the presence or absence of BDM. The loss of dystrophin preceded uptake of the membrane-impermeable Evans blue dye by myocytes that occurred after the withdrawal of BDM and was associated with creatine kinase release and the development of contracture. Although IPC did not alter the redistribution of membrane dystrophin induced by 30 min of ischemia, it facilitated the restoration of membrane dystrophin during reperfusion. Also, myocyte necrosis was not observed when BDM was withdrawn after complete restoration of membrane dystrophin. These results demonstrate that IPC-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contractile force-induced myocardial injury and suggest that the cardioprotection conferred by IPC can be enhanced by the temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion.     

Induction of heat shock protein 72 in the failing heart is attenuated after an exposure to heat shock

Induction of heat shock protein (Hsp) 72 in the right ventricular muscle of the rat with heart failure following acute myocardial infarction (AMI) was examined. AMI was induced by the left coronary artery ligation (CAL). The animals at the 8th, but not 2nd, week after CAL revealed a decrease in cardiac output index (COI), suggesting that heart failure had developed by 8 weeks after CAL. Increases in the right ventricular developed pressure and the ratios of right ventricle/body weight and lung/body weight at the 2nd and 8th weeks showed the development of the right ventricular hypertrophy. After measurement of hemodynamic parameters, the hearts isolated from animals at the 2nd and 8th weeks after CAL (2w- and 8w-CAL hearts, respectively) were perfused and subjected to heat shock (at 42 degrees C, for 15 min) followed by 6-h perfusion. At the end of perfusion, Hsp72 content in the left ventricle without infarct area (viable LV) and the right ventricle (RV) was determined by the Western immunoblotting method. The production of myocardial Hsp72 in the viable LV and RV of the 2w-CAL heart increased after an exposure to heat shock. In contrast, induction of Hsp72 in the viable LV and RV of the 8w-CAL heart was blunted. The results suggest that the development of heart failure following AMI may result in a decrease in the ability for Hsp72 induction not only in the viable LV but also in the RV, leading to contractile dysfunction of the heart.     

Cellular redistribution of inducible Hsp70 protein in the human and rabbit heart in response to the stress of chronic hypoxia: role of protein kinases,

Many infants who undergo cardiac surgery have a congenital cyanotic defect where the heart is chronically perfused with hypoxemic blood. Infant hearts adapt to chronic hypoxemia by activation of intracellular protein kinase signal transduction pathways. However, the involvement of heat shock protein 70 in adaptation to chronic hypoxemia and its role in protein kinase signaling pathways is unknown. We determined expression of message and subcellular protein distribution for inducible (Hsp70i) and constitutive heat shock protein 70 (Hsc70) in chronically hypoxic and normoxic infant human and rabbit hearts and their relationship to protein kinases. In chronically hypoxic human and rabbit hearts message levels for Hsp70i were elevated 4- to 5-fold compared with normoxic hearts, Hsp70i protein was redistributed from the particulate to the cytosolic fraction. In normoxic infants Hsp70i protein was distributed almost equally between the cytosolic and particulate fractions. Hsc70 message and subcellular distribution of Hsc70 protein were unaffected by chronic hypoxia. We then determined if protein kinases influence Hsp70i protein subcellular distribution. In rabbit hearts SB203580 and chelerythrine reduced Hsp70i message levels, whereas SB203580, chelerythrine, and curcumin reversed the subcellular redistribution of Hsp70i protein caused by chronic hypoxia, with no effect in normoxic hearts, indicating regulation of Hsp70i message and subcellular distribution of Hsp70i protein in chronically hypoxic rabbit hearts is influenced by protein kinase C and mitogen-activated protein kinases, specifically p38 MAPK and JNK. We conclude the Hsp70 signal transduction pathway plays an important role in adaptation of infant human and rabbit hearts to chronic hypoxemia.     

Expression and distribution of heat shock proteins in the heart of transported pigs

The expression and localization of four heat shock proteins (Hsp70, Hsp86, Hsp90, and Hsp27) were shown in the heart tissue of pigs transported for 6 h. Immunostaining detected the consistent presence of all Hsps in the pig myocardial cells under both transported and normal housing conditions. Immunohistochemical analysis revealed predominance of Hsp70 (significantly highest levels) and Hsp27 in the cytoplasm of myocardial cells. Hsp90 and Hsp86 were expressed both in the cytoplasm and in the nucleus, preferentially in the cytoplasm, of the myocardial cells. In view of their abundant and uniform distributions in the myocardial cells, the expression and distribution patterns of all detected Hsps within the myocardial cells, mostly limited to the cytoplasm, could be related to their chaperone function for cells with important special activities in this study. The identification of all four Hsps in the blood vessel endothelial cells possibly implies that endothelial cells react to ischemia and hypoxia by expressing Hsps. Immunoblot findings suggest that the level of all Hsps decreased in response to stress due to a 6 h journey. The decrease in Hsp levels in the myocardial cells may indicate that the transport stress may have overcharged the repair mechanisms of the cells. Whether this distinct depletion of Hsps contributes to an increased susceptibility to acute heart failure and the sudden death syndrome in transported pigs should be elucidated in future experiments.