Pharmacological and biological antiviral therapeutics for cardiac coxsackievirus infections

Subtype B coxsackieviruses (CVB) represent the most commonly identified infectious agents associated with acute and chronic myocarditis, with CVB3 being the most common variant. Damage to the heart is induced both directly by virally mediated cell destruction and indirectly due to the immune and autoimmune processes reacting to virus infection. This review addresses antiviral therapeutics for cardiac coxsackievirus infections discovered over the last 25 years. One group represents pharmacologically active low molecular weight substances that inhibit virus uptake by binding to the virus capsid (e.g., pleconaril) or inactivate viral proteins (e.g., NO-metoprolol and ribavirin) or inhibit cellular proteins which are essential for viral replication (e.g., ubiquitination inhibitors). A second important group of substances are interferons. They have antiviral but also immunomodulating activities. The third and most recently discovered group includes biological and cellular therapeutics. Soluble receptor analogues (e.g., sCAR-Fc) bind to the virus capsid and block virus uptake. Small interfering RNAs, short hairpin RNAs and antisense oligonucleotides bind to and led to degradation of the viral RNA genome or cellular RNAs, thereby preventing their translation and viral replication. Most recently mesenchymal stem cell transplantation has been shown to possess antiviral activity in CVB3 infections. Taken together, a number of antiviral therapeutics has been developed for the treatment of myocardial CVB infection in recent years. In addition to low molecular weight inhibitors, biological therapeutics have become promising anti-viral agents.     

Evaluation of antioxidant and immunity function of tetramethylpyrazine phosphate tablets in vivo

The aim of the study was to determine the effect of tetramethylpyrazine phosphate tablets (TPT), a Chinese medicine used for cardiovascular disease, on immunity activity and oxidative injury in rats. Heart failure (HF) was induced by isoproterenol (ISO). After the animal model was established, the rats were administered the TPT by gavage (once a day). The results indicated that TPT improved left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), ±dP/dt, heart weight/body weight. TPT could decrease the levels of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Furthermore, it also could raise the activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), but reduce malonyldialdehyde (MDA) level. The results indicated that TPT improved cardiac function and myocardial fibrosis from myocardial injury, and this cardioprotection might be attributed to a reduction of oxidative stress and regulation of inflammation mediators.     

Morphological and biochemical abnormalities in hearts of cardiac mutant salamanders (Ambystoma mexicanum).

The effect of homozygosity for recessive gene c in Ambystoma mexicanum is the absence of a heartbeat even though initially heart development appears normal. Mutant embryos (c/c) are first distinguishable from their normal siblings (+/+;+/c) at stage 34 (7 days after fertilization) when the normals develop contracting hearts. The mutant hearts at this stage, upon gross examination, appear structurally normal but fail to beat. Nevertheless, the mutants survive through stage 41, which is about 20 days beyond the heartbeat stage, and they exhibit normal swimming movements, indicating that gene c does not affect skeletal muscle. Electron microscopic studies of normal hearts show some myofibrils to be present at stage 34; by stage 41, the normal myocardial cells have become highly differentiated muscle cells. Although some mutant heart cells contain a few thin 60 A and thick 150 A filaments, organized myofibrils are absent. Instead, amorphous proteinaceous collections are prominent. Heavy meromyosin (HMM) binding experiments were performed on mutant hearts to determine whether the myocardial cells contain actin. Mutant myocardial cells that are glycerinated but not treated with HMM contain intact amorphous bodies. After incubation in HMM, the amorphous collections are no longer present and large numbers of decorated actin filaments appear. The.results suggest that the amorphous proteinaceous collections contain actin in a nonfilamentous form, and the addition of HMM induces this actin to polymerize into filaments. SDS-polyacrylamide gel electrophoresis of mutant heart tissue supports this conclusion by showing a prominent 43,000 dalton band suggestive of actin. The electrophoresis experiments also demonstrate a significant reduction of myosin heavy chain (200,000 daltons) in mutant hearts when compared to normal, and this latter observation is confirmed by radioimmunoassay experiments. Muscle tropomyosin (34,000 daltons), prominent in normal hearts, is virtually nonexistent in mutants. Thus, it appears that this single gene mutation affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.     

Changes in expression of cell cycle regulators after G1 progression upon repetitive thioacetamide treatment in rat liver

Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).     

NMR-Based Metabolomic Analysis of Cardiac Tissues Clarifies Molecular Mechanisms of CVB3-Induced Viral Myocarditis and Dilated Cardiomyopathy

Viral myocarditis (VMC), which is defined as inflammation of the myocardium with consequent myocardial injury, may develop chronic disease eventually leading to dilated cardiomyopathy (DCM). Molecular mechanisms underlying the progression from acute VMC (aVMC), to chronic VMC (cVMC) and finally to DCM, are still unclear. Here, we established mouse models of VMC and DCM with Coxsackievirus B3 infection and conducted NMR-based metabolomic analysis of aqueous metabolites extracted from cardiac tissues of three histologically classified groups including aVMC, cVMC and DCM. We showed that these three pathological groups were metabolically distinct from their normal counterparts and identified three impaired metabolic pathways shared by these pathological groups relative to normal controls, including nicotinate and nicotinamide metabolism; alanine, aspartate and glutamate metabolism; and D-glutamine and D-glutamate metabolism. We also identified two extra impaired metabolic pathways in the aVMC group, including glycine, serine and threonine metabolism; and taurine and hypotaurine metabolism Furthermore, we identified potential cardiac biomarkers for metabolically distinguishing these three pathological stages from normal controls. Our results indicate that the metabolomic analysis of cardiac tissues can provide valuable insights into the molecular mechanisms underlying the progression from acute VMC to DCM.     

Analysis of virus protein heterogeneity among group B coxsackie viruses using a "mini" two-dimensional gel electrophoresis system

The application of a small format two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) system to the study of protein heterogeneity among group B coxsackie virus (CVB) isolates is described. Under the conditions of electrophoresis developed during this study, protein samples could be processed within 7 h and up to 300 intracellular proteins were resolved from uninfected HEp-2 cell lysates. 2D-PAGE was used to characterise the intracellular proteins of clinical CVB isolates of serotypes 4 and 5. Intracellular proteins from virus-infected cells were radiolabelled using a pulse-chase protocol under conditions which promoted inhibition of cellular protein synthesis. Depending on the CVB serotype up to 11 intracellular virus proteins were identified, ranging in molecular weight between 14,000 and 54,000. Although the overall two-dimensional protein profiles were characteristic for the two CVB serotypes, within a CVB serotype there was some heterogeneity of the virus proteins, mainly affecting the proteins' net charge. The sensitivity of 2D-PAGE in detecting subtle differences in virus proteins combined with the convenience of the small gel format makes this a suitable approach for the study of the molecular epidemiology of human virus pathogens.     

Evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats

The present study was designed to evaluate the effect of quercetin on myocardial oxidative stress and immunity function impairment induced by isoproterenol in rats. To induce myocardial ischemia, Wistar rats were subcutaneously injected with isoproterenol (70 mg/kg). Blood immunity index, cardiac marker enzymes and antioxidative parameters in hearts were measured. It was found that the levels of blood AST, creatine kinase, NO, NOS, IL-10, IL-1, IL-8 and lactate dehydrogenase in isoproterenol-treated rats were significantly increased. The rats administrated with isoproterenol showed the declines in myocardial antioxidant enzymes activities. Administration of quercetin significantly ameliorated myocardial oxidative injury and immunity function impairment induced by isoproterenol. The results indicated that quercetin possesses activity against isoproterenol-induced myocardial oxidative injury and immunity function impairment, and that the mechanism of pharmacological action was related at least in part to the antioxidant activity of quercetin.     

Local release polymeric-controlled immunotherapy of cardiac transplants in rats

Systemic immunosuppression frequently results in severe side effects. To evaluate a method of limiting the adverse effects of immunosuppression, we implanted controlled release matrices containing cyclosporine-A (Cy) embedded (0.2 or 1 mg/kg/day released), steroid embedded (2%, 0.2% and 0.02% dexamethasone, Dex) or both (Cy 0.2 mg and Dex 0.2%) locally around the transplanted heart at the time of rat heterotopic (neck) heart transplants. Controls received empty (non-drug) matrix implants. To elucidate a local effect, additional groups received Cy (0.2 mg) or Dex (0.2%) matrix implanted in a subdermal distal leg pouch at the time of heart transplant, without a local neck implant. Rejection was determined by the lack of transplanted heart contractions. Recipient animals received no other form of immunosuppression. The Cy (0.2 mg) animals had whole-blood Cy levels monitored for 6 weeks following transplantation. Cy levels peaked at 7–10 days after transplant (119 ± 26 ng/ml) and decayed to <50 ng/ml by day 42. At no time did whole-blood Cy levels reach clinically significant levels. Additional animals had whole-blood, heart and kidney Cy levels measured at day 6 post-transplant. Both doses of local Cy demonstrated good survival benefit and were well absorbed locally, resulting in high Cy levels in heart tissue (>9,000 ng/mg). Furthermore, while low-dose Cy (0.2 mg) demonstrated significant survival benefit, these animals had clinically negligible blood Cy levels on day 6 (<100 ng/ml) and very low kidney Cy levels. Interestingly, the lowest dose of Dex demonstrated no survival benefit, while the mid- and high-Dex doses demonstrated good survival benefit: however, the high-Dex dose had poor wound healing. Cy and Dex combination did not increase efficacy, perhaps due to release problems from physicochemical interactions. Local immunosuppression with a controlled release matrix resulted in a significant survival advantage and was effective in delaying rejection. This approach may prove advantageous clinically, in extending transplantation and lessening immunosuppression side effects.     

Two-dimensional electrophoresis of heart muscle proteins in human cardiomyopathies

Human heart muscle proteins have been analyzed by two-dimensional electrophoresis. Twenty five autopsy heart muscle samples obtained from individuals who had died in accidents and who had no signs of cardiovascular pathology have been compared with biopsy and autopsy myocardium samples of patients with: dilated cardiomyopathy (5 cases), hypertrophical cardiomyopathy (2 cases) and myocarditis (2 cases). In dilated cardiomyopathy in 3 out of 5 cases an additional protein spot was found in the myocardial myosin light chain 1 area.     

Exposure to ultraviolet radiation enhances mortality and pathology associated with influenza virus infection in mice

Ultraviolet radiation (UVR) causes systemic immune suppression, decreasing the delayed type and contact hypersensitivity responses in animals and humans and enhancing certain mycobacterial, parasitic and viral infections in mice. This study tests the hypothesis that prior exposure to UVR enhances influenza infections in mice. BALB/c female mice were exposed to 0-8.2 kJ/m2 of UVR. Exposed and unexposed mice were infected intranasally three days later with 150-300 plaque-forming units/mouse (lethal dose (LD)20-LD40) of mouse-adapted Hong Kong Influenza A/68 (H3N2) virus or sham infected with 50 microL Hanks' balanced salt solution/mouse. Mortality from viral infection ranged from 25-50%. UVR exposure increased virus-associated mortality in a dose-dependent manner (up to a two-fold increase at 8.2 kJ/m2). The increased mortality was not associated with bacterial pneumonia. The highest dose of UVR also accelerated the body weight loss and increased the severity and incidence of thymic atrophy associated with influenza infection. However, UVR treatment had little effect on the increase in lung wet weight seen with viral infection, and, to our surprise, did not cause an increase in virus titers in the lung or dissemination of virus. The mice died 5-6 days after infection, too early for adaptive immune responses to have much impact. Also, UVR did not interfere with the development of protective immunity to influenza, as measured by reinfection with a lethal challenge of virus. Also, cells adoptively transferred from UVR or untreated mice were equally protective of recipient mice challenged with a lethal dose of virus. The mice resemble mice succumbing to endotoxin, and influenza infection increased the levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid and serum cortisol levels; however, UVR preexposure did not increase either of these responses to the virus. The results show that UVR increased the morbidity, mortality and pathogenesis of influenza virus in mice without affecting protective immunity to the virus, as measured by resistance to reinfection. The mechanism of enhanced mortality is uncertain, but the data raises concerns that UVR may exacerbate early responses that contribute to the pathogenesis of a primary viral infection.     

A microfluidic bioreactor for increased active retrovirus output

Retroviruses are one of the most commonly used vectors in ongoing gene therapy clinical trials. To evaluate and advance virus production on the microscale platform, we have created a novel microfluidic bioreactor for continuous retrovirus production. We investigated the growth kinetics of a retroviral packaging cell line in microfluidic bioreactors for several compartment sizes, and packaging cells perfused in the microdevices showed similar growth kinetics to those cultured in conventional static conditions. To evaluate the efficiency of retrovirus production, virus titers from the microdevices were compared to those obtained from static tissue culture. When retrovirus production and collection were maintained at 37 degrees C, virus production levels were comparable for the microdevices and static tissue culture conditions. However, immediate cold storage downstream of the packaging cells in the microdevices resulted in 1.4- to 3.7-fold greater active virus production levels with the microdevices compared to the conventional static conditions over a 5 day period. Lastly, the use of microfluidics for virus production provides a continuous supply of virus supernatant for immediate infection of target cells or for preservation and storage. Such devices will be valuable for the optimization of production and evaluation of retroviruses and other viral vectors for gene therapy applications.     

Monitoring of impending myocardial damage after pleuropneumonectomy and intraoperative photodynamic therapy for malignant pleural mesothelioma using biochemical markers

In five patients who were treated for malignant pleural mesothelioma (MPM) with pleuropneumonectomy and intraoperative photodynamic therapy (IPDT), impending myocardial damage was monitored using ECG, the classical biochemical markers (creatine kinase [CK], total activity; CKMB, mass; and myoglobin), and the new cardiac markers troponin I (cTnI) and troponin T (cTnT). In the peroperative and postoperative period all classical markers were elevated, in contrast to cTnI and cTnT, because of the concomitant skeletal muscle damage. Sequential electrocardiogram monitoring showed no signs of myocardial damage. From this study in patients with MPM treated with pleuropneumonectomy and IPDT it can be concluded that measurement of cTnI and cTnT for the detection of myocardial damage is more suitable than measurement of the classical markers.     

A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue

A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g⁻¹ and 0.15 mg g⁻¹, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.     

Synthesis and antiviral evaluation of novel N-6 substituted adenosine analogues

A series of adenosine analogues were synthesized and their biological evaluation was tested against Coxsackie virus B3 (CVB3) and Herpes simplex virus type 1(HSV-1) in HEp-2 cells. The hydrophobic constant, acute toxicity, carcinogenicity and mutagenicity were calculated. Analogues with piperazine derivatives 8b showed promising activities against CVB3 with a lower IC₅₀value and higher selectivity index, their efficacy was better than that of the commercialized medicine, Ribavirin. These described adenosine analogues exhibit potent antiviral activities against several viruses, and offer new leads for further development.     

Cardioprotective Potential of Garlic Oil and Its Active Constituent,Diallyl Disulphide,in Presence of Carvedilol during Chronic Isoprenaline Injection-Mediated Myocardial Necrosis in Rats

In isoprenaline (ISO)-induced myocardial infarcted rats, garlic oil (GO) and its main ingredient, diallyl disulfide (DADS), were examined for cardioprotective effects when used with carvedilol (CAR). GO, DADS and CAR were given to rats in their respective groups, either alone or together, with the addition of isoprenaline (3 mg/kg/day, subcutaneously) during the last 10 days of treatment. At the end of 14 days of treatment, blood samples were collected, the hearts were excised under anesthesia and weighed. Heart tissue homogenate was used to measure superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid reactive substances (TBARS). Furthermore, the serum activities of cardiac markers, including lactate dehydrogenase, creatine kinase, and cardiac troponin, were checked. Moreover, inflammatory markers including tumor necrosis factor alpha, interleukin one beta, interleukin six, and kappa bp65 subunit were assessed. Rats that received GO, DADS, and CAR exhibited a significant increase in the cardiac antioxidant enzyme activities with a simultaneous decrease in serum cardiac markers enzymes and inflammatory markers. The TBARS were significantly reduced in rats that received treatment. The addition of carvedilol to GO or DADS significantly elevated antioxidant activities and decreased the release of cardiac enzymes into blood circulation. Both DADS and GOl were almost similar in efficacy, indicating the potential role of DADS in garlic oil-mediated cardioprotection. Combining GO or DADS with CAR increased CAR’s cardioprotective impact and protected rats from developing ISO-induced myocardial infarction.     

Comprehensive metabonomic analysis of heart tissue from isoproterenol‐induced myocardial infarction rat based on reversed‐phase and hydrophilic interaction chromatography coupled to mass spectrometry

We aim to describe the metabonomic characteristics of myocardial infarction rats. High‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was utilized to develop a metabonomic method of the heart homogenates of myocardial infarction rats. Hydrophilic interaction chromatography allows the analysis of high polar metabolites, providing complementary information to reversed‐phase liquid chromatography. We combined reversed phase and hydrophilic interaction chromatographic separations to analyze 18 samples, ten from myocardial infarction rat hearts and eight from normal rat hearts. A total of 16 potential biomarkers in rat heart tissue were screened out, primarily related to oxidative stress, nitric oxide damage, taurine, and hypotaurine metabolism and sphingolipid metabolism. This research showed that a comprehensive metabonomic study is a useful tool to reveal the underlying mechanism of myocardial infarction.     

THE LATE PHASE OF HEMATOPORPHYRIN DERIVATIVE-INDUCED PHOTOTOXICITY IN MICE: RELEASE OF HISTAMINE and HISTOLOGIC CHANGES

This study was designed to directly examine the role of mast cells and the histologic changes in the late phase (4-48 h) of hematoporphyrin derivative-induced phototoxicity. BALB/c mice were rendered phototoxic by intraperitoneal injection of HpD, followed by exposure to 1.59 kJ/m2 of 396-406 nm radiation. Immediately before radiation, and at 4, 8, 12, 18, 24 and 48 h after radiation, the ear thickness, serum histamine levels and histologic changes of ears were examined. A maximal net increase in ear thickness of 33.5 +/- 0.3 X 10(-2) mm (mean +/- SE) was noted at 12 h, associated with a maximal net increase of serum histamine (43.3 +/- 11.6 ng/ml, mean +/- SE), and a maximal mast cell degranulation. Other histologic changes consisted of mild epidermal spongiosis at 18-24 h, and a predominant neutrophilic infiltrate, which peaked at 24 h (211.6 +/- 0.4 cells/mm2). No significant alteration was observed in control mice. These data indicated that mast cells participate in the late phase of HpD-induced phototoxicity in mice.     

A Point-of-Care Immunotest for Bedside Determination of Heart-Type Fatty Acid-Binding Protein

Heart-type fatty acid-binding protein (H-FABP) is a small cytosolic protein abundant in heart muscle cells. It offers great potential as a sensitive biomarker for early diagnosis of acute myocardial infarction (AMI).

Ninety-one patients presented to the Emergency Department suspected of AMI with a median symptom onset of 6 h (IQR 3–20 h), of which 75 (82.4%) had AMI. The diagnostic performance of a point-of-care immunotest for H-FABP was compared with those of cardiac troponin T (cTnT), creatinine kinase MB (CK-MB), and myoglobin. The H-FABP immunotest was found to have a significant better sensitivity than the other markers and a better specificity than myoglobin and CK-MB. The H-FABP Immunotest gave the greatest area under the receiver operating characteristic (ROC) curve (0.864) for those admitted within 6 h after the onset of symptoms; whereas, cTnT gave the greatest area under the ROC curve (0.936) for those admitted 6–24 h. The H-FABP was also found to be the most efficient marker to diagnose patients suspected of AMI without ST-elevation and with a negative cTnT.

Early detection of H-FABP using the point-of-care immunotest in patients suspected of AMI may allow more accurate targeting of appropriate therapy and considerable cost savings than the current diagnostic tests.     

Monitoring of water content and water distribution in ischemic hearts

We determined water content and water distribution by fitting dielectric spectra of ischemic canine hearts between 5 MHz and 3 GHz with a newly developed model which describes heart cells and subcellular organelles as rotational ellipsoids filled with electrolyte enclosed by an isolating membrane. The fraction of dry material is modelled by spherical particles with a small dielectric permittivity. Free model parameters were water content, cell volume fraction, and the conductivity of the electrolytes. Resulting model parameters were compared to data from tissue desiccation and to conductivity changes produced by protons and lactate ions. We investigated hearts in two states: during ischemia after interruption of blood flow (pure ischemia, PI, n=5) and during ischemia after resuscitation with Tyrode's solution (IAR, n=14).The difference between water content determined by tissue desiccation and by dielectric spectroscopy was less than 0.5%. During 360 min of ischemia, water content in IAR decreased from 85+/-1.6% to 83+/-2.2% and in PI from 80+/-0.8% to 78+/-1.5%. Cellular volume fraction in IAR increased from 0.47+/-0.045 to 0.63+/-0.031 and in PI from 0.62+/-0.014 to 0.73+/-0.013, which is consistent with published morphometric data. After 180 min of ischemia, the increase of the cytosolic conductivity was 0.14+/-0.02 S/m as calculated from the dielectric spectrum and was similar to the conductivity increase which was roughly estimated on the basis of tissue lactate concentration.In conclusion, dielectric spectroscopy combined with our model analysis facilitates the monitoring of water content and distribution by means of nondestructive surface probes.     

Comparison of two-dimensional electrophoresis patterns of heat shock protein Hsp27 species in normal and cardiomyopathic hearts

Heat shock protein Hsp27 occurs in a complex pattern in human myocardial tissue. Normal and failing explanted human heart from patients with dilated cardiomyopathy (DCM) or ischemic heart failure (IHF), respectively, were analyzed by high resolution two-dimensional electrophoresis (23x30 cm) and Hsp27 immunostaining. Twelve Hsp27 spots in DCM samples were significantly altered in intensity and ten of these were significantly changed in IHF. Four spots (h1, h2, h4, h5) in DCM samples and three spots (h2, h4, h5) in IHF at a molecular mass of 28 kDa were decreased in intensity. In this study, investigating left ventricles of human myocardium, spot h4 was only detected in normal heart samples. On the other hand, spots with a lower molecular mass of 27 kDa (h14, h15, h17, h20, h21) and 22-23 kDa (46, h47, h50) increased in intensity in failing hearts, suggesting that some form of Hsp27 degradation occurs during heart failure.