Heat Shock Protein-70 is expressed in Higher Quantities under Thermal Stress Conditions in Longevity Individuals

Venous blood samples were collected from 54 longevity individuals, with their ages in the range of 90 to 100 years. Among the longevity individuals 35 were males and 20 were females (Table I). The only centenarian individual found was male. Blood samples of non-longevity individuals having the ages in the range of 20-45 years (Table I) were also collected as control. Approximately 3 ml blood was taken from each individual in corning tubes. For serum collection, blood samples were centrifuged at 3000xg for 15 min. Serum in the form of supernatant was collected in a separate tube and stored at -20°C to carry out the assay. Serum Hsp70 levels was determined using sandwich ELISA technique (Elascience; Human HSP-70/HSPA9). The sera were accordingly analyzed using the classical sandwich enzyme-linked immunosorbent assay (ELISA) method as described earlier (Rea et al., 2001; Pockley et al., 2000) with slight modifications. Shortly, stored sera were thawed and the total protein content was quantitatively determined through Bradford method using bovine serum albumin as a standard. The serum was then treated with 0.9% Triton X-100 for 15 min at 15°C with gentle shacking for releasing trapped Hsp70 (Gastpar et al., 2005; Lancaster et al., 2005). The samples were then put onto 96-well microplate (Nunc Immunoplate Maxisorp; Life Technologies) that had been coated with murine monoclonal antihuman Hsp70 (clone C92F3A-5; StressGen) in carbonate buffer, pH 9.2 (2.5 μg/mL) for 24 h at 15°C on gentle agitation, and then washed with PBS containing 1.5% Tween 20 (PBS-T). Blocking was then done by incubating with 1.5% bovine serum albumin in PBS-T. Detection of Hsp 70 was done by adding rabbit polyclonal anti-Hsp70 antibody (SPA-812; StressGen) and this bound polyclonal antibodies were detected with alkaline phosphatase-conjugated murine monoclonal antibody to rabbit immunoglobulins, followed by p-nitro phenyl-phosphate substrate (Sigma Aldrich). The resultant absorbance was measured at 405 nm with a BioRad Benmark Plus plate reader, and the concentrations (ng/ml) of Hsp70 were determined by reference to standard curves (BIOSOFT).

It was hypothesized that hot environmental condition of District D.I. Khan, KP, Pakistan can induce over expression of Hsp70 in longevity individuals. To investigate the hypothesis, serum Hsp70 concentration levels were determined in longevity individuals with their ages in the range of 90-100 years. The same protein was quantitatively measured in non-longevity individuals having ages in the range of 20-30 years as control. The mean concentration among the individuals of 90 year, 91, 92 and 93 years of age were 38.37, 38.35, 37.74 and 38.23 ng/ml respectively, while those of 94 years, 95 years, 96 years, 97 years and 98 years were 40.01, 38.83, 37.42 and 35.99 ng/ml. Interestingly, the only centenarian had comparatively smallest concentration of 28.3 ng/ml among all the longevity individuals (Table I). Maximum concentration of 42.1 ng/ml was found in a male and a female with 95 years and 94 years of age, respectively while the minimum concentration of 28.3 ng/ml was recorded in centenarian male (Table I). Among the non-longevity, the BS zoology and MPhil/PhD students, the highest serum concentration of 38.23 ng/ml of Hsp70 was found in a 26 years old student while the lower quantity of 11.49 ng/ml was recorded in a 19 years old male BS student (Table I). Comparing age-wise mean concentration among the longevity individuals, no significant difference was found. Similarly, there was no significant difference when the mean concentration of the protein was compared between male and female longevity individuals (Table I). In case of non-longevity individuals, the smaller mean concentrations of Hsp 70 were recorded in individuals with age 19. Interestingly, gradual increase in the mean concentration was observed from 20 years of age to 30 years (Table I).

The idea behind quantitative measurements of Hsp70 protein in longevity individuals, in comparison with non-longevity individuals in adverse and continuous heat-stress environmental conditions of D.I. Khan division of southern belt of KP for more than nine months in a year, was see any possible influence of the protein on longevity. Previous findings regarding the relationship of extracellular Hsp70 with aging and longevity were heterogeneous. In an earlier report (Rea et al., 2001) conducted on individuals having their ages in the range of 20 to 96 years, inverse relationship between serum Hsp70 and age was observed as the protein had progressive decrease with age. Similarly, in another finding conducted on Chinese males of 15 to 50 years of age, the same progressive decline of serum Hsp70 with age was reported but a positive correlation of the protein with age was also observed in younger individuals (Jin et al., 2004). These findings hence report decline in serum Hsp70 levels as human ages but this decline within an individual over time has not been indicated, although in vitro experiments on cell lines of centenarians showed increased expression of Hsp70 against heat stress conditions (Ambra et al., 2004), showing the protein as a marker of health, indicator of stress (Jin et al., 2004; Ambra et al., 2004). Another possibility of low concentration of serum Hsp70 shown in earlier report (Njemini et al., 2006) was the use of modified form of commercially available kit that was detecting comparatively lower concentration of the protein because of washing the samples with Triton X-100. Here, the findings of the present study are totally in contrast and in disagreement with what has previously been described. The mean concentrations of Hsp70 in longevity individuals, both males and females were comparatively significantly higher than non-longevity individuals. No significant difference was found in the mean concentrations between mael and female longevity individuals. Interestingly, the means values had increase with increase in age in non-longevity individuals. This significant difference between the present study and previous studies can be due the reasons: (i) continued heat stress conditions in D.I. Khan division throughout the year, (ii) life style of people especially longevity individuals, living in the remote areas in the villages, working hard in the field throughout the year, lack even the basic health facilities and thus never rely on medicines, (iii) possibly, inheritance factor is also contributing to some extent as 70% the non-longevity individuals possessing comparatively higher concentration of Hsp70 belong to the family of longevity individuals, (iv) increased synthesis of Hsp70 protein in longevity individuals seems to have compensating influence against diseases, a signal molecule and a marker of good health. The protein probably acts more than a chaperon in these individuals, stimulate the adaptive immune response and regulate other biochemical processes vital for healthy long life, and (v) it has been shown that low concentration of Hsp70 is directly related to decreased exposure to inflammation and inflammation has influence on cardiovascular diseases ( Pockley and Frostegard, 2005). But interestingly during this study, the longevity individuals had comparatively higher concentrations of Hsp70 and nobody was found to have cardiovascular diseases that needs to be deciphered.

Increased synthesis of Hsp70 protein in longevity individuals seems to have compensating influence against diseases, a signal molecule and a marker of good health. The protein probably acts more than a chaperon in these individuals.

The authors have declared no conflict of interests.