INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

SEVERE MYOCARDIAL ISCHEMIA results in irreversible injury of myocytes, which is manifest classically as coagulation necrosis. Despite many years of active research, the exact series of subcellular events underlying the transition from reversible to irreversible injury remains elusive. It is known that certain interventions are capable of modulating or delaying the onset of irreversible injury in experimental model systems such as hypothermia (1, 15), calcium channel blockade (19, 28, 36), and more recently, ischemic preconditioning (16, 26, 29, 32). However, even in experimental model systems, the mechanisms responsible for protection are not fully known. Previous studies have shown that increased expression of heat shock proteins (HSPs), specifically HSP70i, protects ischemic myocardium. Protection with HSP70i against ischemic injury has been shown in a variety of model systems including myogenic cell lines (9), neonatal cardiomyocytes (24), transgenic mice overexpressing HSP70i (23), and whole animals (4, 22). However, heat shock per se (rapidly raising the core temperature of the animal above the normal body temperature) and/or chemical stimulators of HSPs may activate several other members of the large HSP family, which may also be cardioprotective. Such a result would complicate interpretation of the role induction of any specific HSP plays in the mechanism of cardioprotection.

Small HSPs (sHSPs) are a subgroup of the HSP family. Certain members of the sHSP family, specifically HSP27 and -crystallin, have several biological characteristics that render them excellent candidates for playing an important role in cardioprotection. For example, sHSPs such as HSP27 and -crystallin are known to exist in measurable quantities in mammalian myocardium and are induced by oxidative stress, an important component of ischemia-reperfusion (I/R) injury (3). In addition, sHSPs are known to function as "chaperone proteins," proteins that work to facilitate protein folding and translocation at the subcellular level (5). sHSPs are also known to be associated with cytoskeletal proteins. This association may be important for two reasons. First, HSP27 has been shown in simple cellular systems to stabilize certain cytoskeletal structures, which in turn have been associated with increased resistance to stress (8, 11). Second, because irreversible ischemic injury in myocardium is thought to involve critical lesions to the cytoskeletal support system (7, 12, 30, 34), proteins that could bind to and protect these critical proteins would be predicted to protect against lethal cell injury. Indeed, sHSPs have been shown to protect against a variety of lethal injurious insults including hyperthermia, toxic chemical agents, oxidative injury, and osmotic swelling (2). Martin et al. (24) reported that increased expression of HSP27 and -crystallin provided cardioprotection against hypoxic injury in cultured adult (HSP27) and neonatal (-crystallin) rat myocytes, lending further support to the concept that sHSPs play an important role in lethal ischemic injury.

However, to date no studies investigating the potential cardioprotective role of sHSPs have been conducted in large animal species. Large animal models of myocardial I/R injury are generally considered more applicable to human hearts, because their heart rates and blood pressures are more similar to humans. Furthermore, in vivo studies to date have not resulted in consistent, reproducible overexpression of transgenic proteins. Therefore, it is of considerable interest whether overexpression of potentially cardioprotective proteins, specifically sHSPs, in adult myocytes from a large animal species will result in a cardioprotective effect.

The purpose of the present study was twofold: first, to determine whether isolated adult canine ventricular myocytes could be cultured and infected with a replication-deficient recombinant adenovirus that would result in overexpression of a sHSP without injuring the myocytes; and second, to determine whether increased expression of HSP27 in adult canine myocytes results in a cardioprotective effect in response to simulated I/R injury.

	METHODS AND MATERIALS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

All experiments reported here conformed to the standards in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1985). Adult mongrel dogs (n = 14) of either sex, weighing between 10 and 15 kg, having hematocrits >35, and free from clinically relevant disease, were anesthetized with 25-35 mg/kg pentobarbital sodium administered intravenously. Animals were then intubated and mechanically ventilated (200 ml · kg¹ · min¹; Harvard respirator) using room air supplemented with 100% oxygen.

After a deep plane of anesthesia was achieved, a left thoracotomy was performed in the fourth intercostal space and the heart was rapidly excised and immediately plunged into ice-cold potassium chloride solution to arrest the heart before transport to the laboratory for isolation of the ventricular myocytes.

Preparation and perfusion of the heart. After excision, the heart was weighed and wet weight was recorded for calculation of perfusion rates. The aortic arch was trimmed and opened, and a small segment of Silastic tubing connected to a three-way stopcock was inserted and tied into the proximal aorta. After cannulation, the position of the cannula was checked to ensure that it was distal to the origin of the coronary ostia. The coronary arteries were flushed with 125 ml of ice-cold calcium-free modified Krebs-Henseleit perfusion buffer (KHB) to ensure that all blood was removed from the coronary arteries. The KHB perfusion buffer contained (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.68 glutamine, 11 glucose, 5 Na-pyruvate, 5 NaHCO3, and 20 HEPES; and (in ml/l) 10 basic essential vitamins, 10 minimal essential vitamins, and 10 vitamins.

Isolation of adult canine ventricular myocytes. After cannulation, the heart with the Silastic cannula attached was hung on a modified Langendorff-perfusion apparatus and perfused in a nonrecirculating mode with calcium-free KHB perfusion buffer at 37°C for 10 min at a flow rate of 1 ml · min¹ · g¹ based on the recorded heart wet weight. During all perfusion and subsequent disaggregation steps, the appropriate solutions were continuously bubbled with 95% O₂-5% CO₂. During the calcium-free period, the heart was perfused. After 10 min, the perfusion solution was changed to calcium-free KHB buffer containing 120 mg of type IV collagenase (Worthington; Freehold, NJ). The perfusion mode was switched to recirculating and the collagenase solution was allowed to digest the heart for 25 min (the optimal duration of collagenase perfusion was determined in a series of pilot experiments). After 25 min of collagenase perfusion, the heart was taken down and the left ventricular outflow tract was opened to expose the endocardial surface of the left ventricular cavity. The endocardium was removed with scissors and the underlying softened left ventricular muscle was harvested, minced with scissors, placed in 80 ml of fresh calcium-free isolation buffer (37°C), and transferred to a shaking water bath operating at 37°C. The isolation buffer was a modified KHB containing (in mM) 80 KCl, 30 KH2PO4, 4 MgSO4, 20 HEPES, 10 glucose, 20 taurine, 5 creatine, 5 succinate, and 0.1% BSA (pH 7.25). The potassium concentration was raised to maintain electrical arrest of the tissue to preserve high-energy phosphates (ATP). The partially digested myocardium was gently agitated in the shaking water bath for 20 min. During this time, the tissue was continuously bubbled with 95% O₂-5% CO₂ gas and was frequently triturated with a Silastic transfer pipette to encourage further disaggregation of the myocytes. After 20 min of incubation, the final digest was filtered through a fine-mesh nylon filter to remove large pieces of collagen and debris.

Purification of myocytes. After completion of the tissue digest, the percentage of rod-shaped cells was increased by gravity-settling the myocytes three times over 2 ml of KHB solution containing 4% BSA. After each settling, the resulting loose pellet was resuspended in fresh KHB buffer containing 0.5% BSA. In pilot experiments, this purification procedure increased the yield of rod-shaped cells ~50% compared with nonsettled cells. After completion of the purification procedure, the cells were pooled and the percentage of rod-shaped cells (vs. round cells) and the overall viability was assessed by mixing an aliquot of the myocytes with Trypan blue (vital dye). A typical isolation yielded ~10 million cells of which 60-80% were rod-shaped. Nearly 100% of the rod-shaped cells were viable (negative stain with Trypan blue), whereas ~95% of the round-shaped cells were nonviable (positive stain with Trypan blue). Therefore, overall viability was approximately 60-80%.

Cell culture. After isolation and purification of the myocytes, the cells were cultured on six-well plates (Corning; Corning, NY) for 1 h before infection. To optimize attachment of the cells, the plates were precoated with laminin 24 h before the myocyte isolation and kept in the tissue culture incubator at 37°C until the day of isolation. After isolation and purification, the myocytes were removed from the isolation buffer and resuspended in a modified tissue culture media [containing medium 199 supplemented with 0.2% albumin, 5 mM taurine, 3.0 mM creatine, 3.0 mM carnitine, 10⁶ M thyroxine, and antibiotics (penicillin-streptozotocin and gentamicin) to inhibit bacterial growth]. After counting the cells using a hemocytometer, ~3.0 ×10⁵ cells were plated in each well of the six-well plates. Cells were allowed to attach for 1 h and then the media was changed with fresh culture media to remove unattached myocytes.

Construction of recombinant adenovirus. The 0.76-kb coding region of the human HSP27 cDNA (provided by Drs. L. Weber and E. Hickey, University of Nevada, Reno, NV) was used to make the construct. The appropriate fragments were cloned between the enhancer/promoter of the cytomegalovirus immediate-early genes and the Simian virus 40 polyadenylation signal of the pACCMV.pLpA shuttle vector provided by Dr R. Gerard. Replication-deficient adenovirus was generated through homologous recombination of two plasmids (pJM17, a bacterial plasmid containing the full-length adenoviral genome, and the shuttle vector) after cotransfection into E1-transformed human embryonic kidney 293 cells to produce E1-deleted adenovirus encoding the appropriate transgene 27. Viral stocks were generated by infecting confluent HEK-293 cells, harvesting the cells, and concentrating the cells through CsCl ultracentrifugation. Viral stocks were then desalted through a Sepharose CL4B (Sigma) column into a Tris-buffered solution, plaque-titered, aliquoted, and stored at 70°C with 10% glycerol until use.

Experimental protocol. Myocytes were divided into three groups: control, experimental virus-infected, and control virus-infected. The experimental group was infected with replication-deficient adenovirus (all adenoviral constructs were a gift from Drs. Ruben Mestril and Jody Martin, Loyola University, Chicago, IL) containing the cDNA of human HSP27. Another group of myocytes from the same isolation was infected with "empty virus"; a similar strain of replication-deficient adenovirus that did not contain a coding sequence. Finally, one group of myocytes was cultured for 3 days without adenovirus infection. After 3 days of incubation, the three groups of myocytes were each split into two equal halves (three wells from each six-well plate). One half of each plate was used for Western blot analysis of HSP27 protein expression and the other half was used to test for cardioprotection from simulated myocardial I/R injury. The culture media was removed from myocytes to be subjected to simulated ischemia and replaced with new media containing 3.0 mM iodoacetic acid (IAA) to inhibit glycolysis and 3.0 mM amobarbital to inhibit mitochondrial respiration (33). Myocytes incubated in the ischemic buffer were monitored for the development of ischemic contracture as indicated by the assumption of a "square" shape (33). After visual confirmation of the onset of ischemic contracture, cells were "reperfused" with fresh oxygenated culture media without the chemical inhibitors for 10 min and the proportion of live and dead cells was determined using Trypan blue exclusion criteria.

Western blot procedures. Half of each six-well plate not subjected to simulated ischemia was harvested for protein analysis using standard Western blot techniques. Briefly, cells were lysed, placed into a standard sample buffer, and subjected to protein electrophoresis using 7.5% gels. Each well was loaded with an equal amount of protein as determined previously using a standard spectrophotometric protein assay. After electrophoresis, the proteins were transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in Tween 20 Tris-base sodium for 2 h at room temperature and then incubated overnight at 4°C with a rabbit polyclonal anti-HSP27 primary antibody (at a dilution of 1:1,000). The next day, the membranes were incubated with a goat anti-rabbit IgG (Transduction Laboratories) at a 1:5,000 dilution, and HSP27 protein expression was detected using a chemiluminescence detection system (Amersham; Arlington, IL).

Statistics. Each myocyte isolate generates control and experimental groups, and therefore, each isolate serves as its own control. Statistically significant differences among groups were tested using a paired t-test analysis. In all analyses, a P value of <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Culture of adult canine ventricular myocytes. We determined that isolated left ventricular myocytes would represent the best model to test whether increased expression of HSP27 would be cardioprotective in canine myocardium. However, to test this hypothesis requires a selective increase of HSP27 expression in ventricular myocytes without compromising viability and without inducing increased expression of HSP27 in other nonmyocyte cells. Because increased protein expression needs time to develop, the first goal of this study was to develop a technique to culture adult canine myocytes and keep them viable for the period of time required to generate increased expression of a transgenic protein. Using techniques previously reported to isolate adult rat ventricular myocytes (33, 35), we are routinely able to isolate large numbers of viable adult canine left ventricular myocytes. Canine myocytes can be kept in culture for up to 6 days without significant loss of viability or dedifferentiation (Fig. 1 shows cells after 3 days of culture). After establishing this technique (details in METHODS AND MATERIALS), we sought to determine whether cultured adult canine ventricular myocytes could be directed to increase a transgenic protein using replication-deficient adenoviral techniques.

View larger version (134K): [in this window] [in a new window]   Fig. 1.   Adult canine ventricular myocytes after 3 days in culture. Myocytes show intact rod shape and show no evidence of dedifferentiation (×250).

Effect of adenoviral infection on adult canine myocytes. It has been reported that infection of neonatal and adult rat myocytes with replication-deficient adenoviral constructs results in increased expression of the transgene (24). However, to date there have been no reports on whether adenoviral techniques are effective in increasing protein expression in adult canine ventricular myocytes. Therefore, one of the primary purposes of this study was to determine the optimal conditions under which adult canine myocytes can be induced to increase expression of a transgenic protein. As shown in Fig. 2, using an adenovirus previously shown to result in a robust increase in HSP27 protein expression in neonatal rat myocytes, infection of adult canine myocytes resulted in a large increase in myocyte HSP27 content (average two- to threefold vs. control cells at 3 days). Importantly, the viability of infected myocytes did not differ significantly from control cells (Fig. 3). After confirming that adult myocytes could be successfully induced to increase expression of HSP27, we sought to determine the optimal dose and time of adenoviral infection. As shown in Fig. 4, myocytes infected with 250 multiplicity of infection (MOI) expressed more HSP27 protein than those infected with 100 MOI. However, increasing the MOI further to 750 did not result in significantly more HSP27 expression. Furthermore, we determined that extending the duration of postinfection culture (at a given MOI) to 6 days did not result in more HSP27 expression than was present at 3 days (Fig. 4). On the basis of these results, the remaining experiments were conducted using myocytes 3 days after infection with adenovirus.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Equal numbers of freshly isolated adult canine myocytes were plated on coated dishes. Cultures of standard HEK-293 cells served as a control. After 1 h, cells were infected with adenovirus designed to increase expression of human heat shock protein 27 (HSP27). After 3 days in culture, cells were harvested and the resulting Western blot probed for HSP27 protein expression. Infection with adenovirus resulted in a large increase in HSP27 expression in both 293 cells and adult canine ventricular myocytes.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Equal numbers of freshly isolated adult canine left ventricular myocytes were plated on laminin-coated flasks. After 1 h, myocytes were infected with adenovirus and were maintained in culture for 3 days. At the time of infection, cultured cells from both groups were 100% viable. After 3 days in culture, both groups showed identical viability (control = 89 ± 1.5% vs. infected = 89 ± 5.1%; n = 3; P = not significant).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Equal numbers of freshly isolated adult canine left ventricular myocytes were plated on laminin-coated flasks. After 1 h, cells were infected with adenovirus at the indicated multiplicity of infection (MOI). Cells were maintained in culture for either 3 or 6 days and then harvested for Western blot analysis of HSP27 protein expression. The amount of HSP27 protein expression increased with increasing dose (MOI) and time of infection. (Representative blot of 5 separate experiments).

Effect of increased HSP27 expression on response to simulated I/R injury. Having established the optimal dose and time course for increased expression of HSP27, we sought to determine whether increased HSP27 expression would protect isolated myocytes against simulated I/R injury. Myocytes were infected with adenovirus for 3 days at 250 MOI and then subjected to I/R. The viability of the myocytes over 3 days of culture did not differ between control and infected myocytes (89.0 ± 1.5% control vs. 89 ± 5.1% adenoviral infected; P = not significant; Fig. 3). Incubating the myocytes with inhibitors of both glycolysis (IAA) and mitochondrial respiration (amytal) simulated I/R injury. From previous studies, it is known that the time course to ischemic contracture (as indicated by the assumption of a "square" shape vs. the normal rod-shaped configuration) is variable among individual myocytes, but the subsequent period of time (from onset of contracture to irreversible injury) is constant (31). The use of the combination of IAA and amytal overcomes this problem by accelerating energy depletion and thereby "synchronizing" the onset of ischemic contracture. In these experiments, myocytes were monitored for the onset of contracture after the addition of IAA and amytal. The time point at which ischemic contracture developed was somewhat variable (range 12-45 min; mean = 19.16 ± 5.8 SE min; n = 6) among individual isolates (reflecting the importance of using each isolate as its own control) but was synchronous for all myocytes on a given culture plate. After the onset of contracture, myocytes were incubated an additional 20 min with IAA plus amytal. After 20 min, the ischemic buffer was removed and control oxygenated culture media was added for 15 min to wash out the chemical inhibitors and thereby simulate reperfusion. After I/R, 34.4% of control myocytes were viable using Trypan blue exclusion criteria, whereas 51.3% of myocytes expressing HSP27 were viable (n = 6). Myocytes infected with the empty virus were not significantly different from control uninfected myocytes (data not shown). Figure 5 summarizes the data and shows that compared with control myocytes, myocytes expressing HSP27 were protected from simulated I/R injury.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Equal numbers of freshly isolated adult canine left ventricular myocytes were plated on laminin-coated flasks. Myocytes were infected with adenovirus at 250 MOI and maintained in culture for 3 days. Myocytes were subjected to simulated ischemia-reperfusion via exposure to chemical inhibitors (CI) followed by resuspension in normal media. Expression of HSP27 resulted in significant protection compared with control cells (P < 0.001; n = 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

The major findings of this study were that 1) adult canine left ventricular myocytes can be isolated and cultured for several days without showing evidence of dedifferentiation or significant loss of viability, 2) cultured adult canine myocytes can be infected with a replication-deficient adenovirus that increases the expression of a transgenic protein without loss of viability or significant alteration in myocyte architecture, and 3) increased expression of the small HSP27 is associated with protection of isolated adult canine myocytes from simulated I/R injury.

Cultured adult canine ventricular myocytes. Several model systems incorporating cultured myocytes have been developed to study various aspects of cardiovascular pathophysiology. Cultured myocytes offer several advantages over whole organ and/or whole animal studies including the ability to study relatively pure populations of myocytes, the ability to precisely control the experimental conditions and the doses of administered pharmacological agents, and the ability to manipulate and measure protein synthesis and production. In addition, cultured myocytes provide an attractive model system for the application of transgenic experimental approaches. For example, to investigate the selective manipulation of targeted intracellular proteins requires that the manipulation occur only in myocytes and not in vascular smooth muscle cell, endothelial cells, or other cellular elements present in whole organs or animals. Furthermore, selective manipulation of intracellular myocyte proteins requires a targeted delivery system. Many proteins exist in large families and physical or chemical upregulation of one family member more often than not results in upregulation of unintended other family members, which complicates interpretation of the resulting data.

Infection of canine myocytes with adenovirus. Previous studies have also shown that transgenic proteins can be expressed using different expression techniques ranging from lipafectamine to direct injection into the myocardium. More recent studies have demonstrated that replication-deficient adenoviral constructs can be useful tools in the selective modulation of intracellular myocyte proteins. For example, the study by Martin et al. (24) showed that expression of the small HSP27 (using the virus utilized in the present study) can be significantly increased in cultured neonatal and adult rat myocytes.

Effect of increased HSP27 expression on the response to simulated I/R. Numerous studies have shown that HSPs, specifically HSP70 and sHSPs such as HSP27, are capable of causing substantial protection from the irreversible injury associated with I/R (2). Protection from I/R injury has been documented in many different model systems ranging from in vitro cell culture to transgenic mice (4, 9, 22-24). However, until recently, most studies have utilized heat shock to increase or induce the expression of HSP. Martin et al. (24) utilized an adenovirus to selectively increase the expression of HSP27, which proved to be protective against simulated I/R injury in adult rat myocytes. The present study was designed to study selective overexpression of HSP27 in adult canine ventricular myocytes. Results show that increased expression of HSP27 results in protection from simulated I/R injury in a separate adult species. The fact that HSP27 has now been shown to protect in two adults species in independent studies further underlines the importance of investigating the mechanism through which sHSPs protect myocytes from I/R injury.

Possible mechanisms of HSP27-induced effect. Many studies have shown that increased expression of HSP results in protection from lethal I/R injury, but the mechanism through which they act remains open to speculation. Stress proteins have the potential to inhibit or reduce lethal injury through many different ways, including inhibition of proapoptotic pathways, increasing expression of antioxidant proteins, inhibiting release of suicide activators of cytochrome c, preventing the destruction of key structural cytoskeletal proteins, or a combinatorial or additive effect (10, 17, 18, 20, 25, 27). Results from recent studies support the notion that sHSPs such as HSP27 may work through many unique, protective pathways. For example, increased expression of HSP27 has been shown to cause an increase in cellular levels of the antioxidant protein glutathione as well as decreased Fas/APO-1-mediated apoptosis (17, 25). On the contrary, reduction of HSP27 expression has been shown to decrease glutathione levels and result in increased rates of apoptosis (17, 25). In addition to affecting apoptotic pathways and antioxidant protein levels, sHSPs have been shown to bind to and reduce the destruction of actin fibers in other cell systems, a function commonly known as "chaperone" (5). Although there is no direct evidence that HSP27 performs a chaperone function in myocytes, it is logical to hypothesize that the chaperone function of sHSPs may also extend to critical cytoskeletal proteins in myocytes. Cytoskeletal proteins are structural proteins that play an important role in maintaining the intact highly ordered structure of the myocyte under normal conditions as well as in response to cellular stress (6). Indeed, evidence that critical lesions develop in the myocyte cytoskeleton before the onset of irreversible injury has existed for many years (6, 7, 12, 30, 34). It is possible that during the early phases of ischemic injury, sHSPs bind to critical cytoskeletal proteins thereby preventing their destruction and delaying the onset of lethal cell injury. If so, then HSP27 could be acting through multiple different cytoskeletal proteins, i.e., talin, dystrophin, vinculin, or spectrin, to accomplish cellular protection.