Effects of heat shock on amino Acid metabolism of cowpea cells

When cowpea (Vigna unguiculata) cells maintained at 26°C are transferred to 42°C, rapid accumulation of γ-aminobutyrate (>10-fold) is induced. Several other amino acids (including β-alanine, alanine, and proline) are also accumulated, but less extensively than γ-aminobutyrate. Total free amino acid levels are increased approximately 1.5-fold after 24 hours at 42°C. Heat shock also leads to release of amino acids into the medium, indicating heat shock damage to the integrity of the plasmalemma. Some of the changes in metabolic rates associated with heat shock were estimated by monitoring the ¹⁵N labeling kinetics of free intracellular, extracellular and protein-bound amino acids of cultures supplied with ¹⁵NH₄⁺, and analyzing the labeling data by computer simulation. Preliminary computer simulation models of nitrogen flux suggest that heat shock induces an increase in the γ-aminobutyrate synthesis rate from 12.5 nanomoles per hour per gram fresh weight in control cells maintained at 26°C, to as high as 800 nanomoles per hour per gram fresh weight within the first 2 hours of heat shock. This 64-fold increase in the γ-aminobutyrate synthesis rate greatly exceeds the expected (Q₁₀) change of metabolic rate of 2.5- to 3-fold due to a 16°C increase in temperature. We suggest that this metabolic response may in part involve an activation of glutamate decarboxylase in vivo, perhaps mediated by a transient cytoplasmic acidification. Proline appears to be synthesized from glutamate and not from ornithine in cowpea cells. Proline became severalfold more heavily labeled than ornithine, citrulline and arginine in both control and heat-shocked cultures. Proline synthesis rate was increased 2.7-fold by heat shock. Alanine, β-alanine, valine, leucine, and isoleucine synthesis rates were increased 1.6-, 3.5-, 2.0-, 5.0-, and 6.0-fold, respectively, by heat shock. In contrast, the phenylalanine synthesis rate was decreased by 50% in response to heat shock. The differential effects of heat stress on metabolic rates lead to flux and pool size redistributions throughout the entire network of amino acid metabolism.     

Interaction of heat and salt shock in cultured tobacco cells

Cultured tobacco cells (Nicotiana tabacum L. var Wisconsin-38) developed tolerance to otherwise nonpermissive 54°C treatment when heat-shocked at 38°C (2 h) but not at 42°C. Heat-shocked cells (38°C) exhibited little normal growth when the 54°C stress came immediately after heat shock and normal growth when 54°C stress was administered 8 hours after heat shock. Heat shock extended the length of time that the cells tolerated 54°C. Tobacco cells developed tolerance to otherwise lethal 2% NaCl treatment when salt-shocked (1.2% NaCl for 3 hours). The time course for salt tolerance development was similar to that of thermotolerance. Heat-shocked cells (38°C) developed tolerance of nonpermissive salt stress 8 hours after heat shock. Alternatively, cells heat-shocked at 42°C exhibited immediate tolerance to lethal salt stress followed by a decline over 8 hours. Radioactive methionine incorporation studies demonstrated synthesis of heat shock proteins at 38°C. The apparent molecular weights range from 15 to 115 kilodaltons with a protein complex in the 15 to 20 kilodalton range. Synthesis of heat shock proteins appeared to persist at 42°C but with large decreases in incorporation into selected heat shock protein. During salt shock, the synthesis of normal control proteins was reduced and a group of salt shock proteins appeared 3 to 6 h after shock. Similarities between the physiology and salt shock proteins/heat shock proteins suggest that both forms of stress may share common elements.     

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes

The effects of heat shock on the synthesis of α-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25°C to 40°C for 3 hours, inhibits the accumulation of α-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca²⁺. When ER is isolated from heat-shocked aleurone layers, less newly synthesized α-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca²⁺ transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

Heat Stress Responses in Cultured Plant Cells : Heat Tolerance Induced by Heat Shock versus Elevated Growing Temperature

Using cultured pear (Pyrus communis cv Bartlett) cells, heat tolerance induced by heat shock was compared to that developed during growth at high temperature. After growth at 22°C, cells exposed to 38°C for 20 minutes (heat shock) showed maximum increased tolerance within 6 hours. Cells grown at 30°C developed maximum heat tolerance after 5 to 6 days; this maximum was well below that induced by heat shock. Heat shock-induced tolerance was fully retained at 22°C for 2 days and was only partly lost after 4 days. However, pear cells acclimated at 30°C lost all acquired heat tolerance 1 to 2 days after transfer to 22°C. In addition, cells which had been heat-acclimated by growth at 30°C showed an additional increase in heat tolerance in response to 39°C heat shock. The most striking difference between heat shock and high growth temperature effects on heat tolerance was revealed when tolerance was determined using viability tests based on different cell functions. Growth at 30°C produced a general hardening, i.e. increased heat tolerance was observed with all three viability tests. In contrast, significantly increased tolerance of heat-shocked cells was observed only with the culture regrowth test. The two types of treatment evoke different mechanisms of heat acclimation.     

Effects of the herbicide san 9789 on photomorphogenic responses

The herbicide, 4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)- 3(2H)-pyridazinone (San 9789), an inhibitor that prevents both carotenoid and chlorophyll accumulation and normal chloroplast development in white light, does not affect the physiological effectiveness of phytochrome in dark-and light-grown plants. Red/far red reversibility of growth inhibition, stimulation of anthocyanin synthesis, and stimulation of phenylalanine ammonia-lyase synthesis are not significantly different in plants grown with and without San 9789. Despite the complete absence of photosynthesis, flowering could be induced in the long day plant Hordeum vulgare L. when sucrose was provided to the leaves. Since the nonphotochemical reactions of phytochrome also are not affected by the herbicide, San 9789 may be used as a tool to study the phytochrome system spectrophotometrically in plants grown for relatively long periods under high intensity white light.     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

Selective stimulation of Hsp27 and αB-crystallin but not Hsp70 expression by p38 MAP kinase activation

The levels of Hsp27 and αB-crystallin in C6 rat glioma cells, that had been heated at 43°C for 30 min with a subsequent culture for 16 h at 37°C, were markedly increased. The exposure of the cells to a low concentration (0.1–3 µg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and αB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and αB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1–3 µg/ml anisomycin. The results of a reporter assay, using an αB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of αB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 µg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO₂, or CdCl₂. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 µg/ml of anisomycin. The heat-induced accumulations of Hsp27 and αB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and αB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and αB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.     

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26°C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42°C. At 34 and 38°C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42°C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38°C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38°C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.     

Acquisition of Thermotolerance in Soybean Seedlings : Synthesis and Accumulation of Heat Shock Proteins and their Cellular Localization

When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28°C up to 40°C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45°C followed by incubation at 28°C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45°C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40°C or a brief (10-minute) pulse treatment at 45°C followed by a 28°C incubation provide protection (thermal tolerance) to a subsequent exposure at 45°C. Maximum thermoprotection is achieved by a 2-hour 40°C pretreatment or after 2 hours at 28°C with a prior 10-minute 45°C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSP-like proteins, and also provides thermoprotection to a 45°C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions.

During 40°C HS, some HSPs become localized and stably associated with purified organelle fractions (e.g. nuclei, mitochondria, and ribosomes) while others do not. A chase at 28°C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40°C chase period. If the seedlings are subjected to a second HS after a 28°C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions. The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40°C to 45°C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28°C but become organelle-associated during a subsequent HS at 40°C.

     

Thermotolerance is developmentally dependent in germinating wheat seed

During the initial 9 to 12 hours of imbibition, the imbibing wheat (Triticum aestivum L.) seed was found to exhibit substantial tolerance to high temperature relative to later times of imbibition. Tolerance was assessed by seed viability and seedling growth. This initial high temperature tolerance gradually declines with increasing time of seed imbibition. A range of 2 hour heat pretreatments (38-42°C) prior to imposition of a 2 hour heat shock (51-53°C) during this same 9 to 12 hour interval was unable to increase survival or seedling growth over that of seed that did not receive a pretreatment. However, after 9 to 12 hours of imbibition the pretreatment provided both increased survival and increased seedling growth, measured 120 hours later, i.e., classical thermotolerance could be acquired. This response is called a `thermotolerance transition.' Isolated embryos responded in a similar manner using a 2,3,5-triphenyltetrazolium chloride assay for viability determination following heat treatments. The high temperature tolerance during early imbibition indicates that the thermotolerance transition involves the loss of an existing thermotolerance coincident with acquiring the ability to become thermotolerant following heat pretreatment. Despite the inability to acquire thermotolerance, heat shock protein synthesis was induced by heat shock immediately upon imbibition of wheat seed or isolated embryos. Developmentally regulated heat shock proteins of 58 to 60, 46, 40, and 14 kilodaltons were detected at 1.5 hours of imbibition following heat shock, but were absent or greatly reduced by 12 hours. Constitutive synthesis of 70 and 90 kilodalton hsp groups appeared to be greater at 1.5 hours of imbibition than at 12 hours of imbibition.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the α- and β- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using ³⁵SO₄ and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t_½ = 50 versus 150 minutes, respectively) and during the deinduction period (t_½ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress.     

A class of soybean low molecular weight heat shock proteins : immunological study and quantitation

Two major polypeptides of the 15- to 18-kilodalton class of soybean (Glycine max) heat shock proteins (HSPs), obtained from an HSP-enriched (NH₄)₂SO₄ fraction separated by two-dimensional polyacrylamide gel electrophoresis, were used individually as antigens to prepare antibodies. Each of these antibody preparations reacted with its antigen and cross-reacted with 12 other 15- to 18-kilodalton HSPs. With these antibodies, the accumulation of the 15- to 18-kilodalton HSPs under various heat shock (HS) conditions was quantified. The 15- to 18-kilodalton HSPs began to be detectable at 35° C, and after 4 hours at 40° C they had accumulated to a maximum level of 1.54 micrograms per 100 micrograms of total protein in soybean seedlings and remained almost unchanged up to 24 hours after HS. Accumulation of the HSPs was reduced at temperatures higher than 40° C. At 42.5° C the HSPs were reduced to 1.02 micrograms per 100 micrograms, and at 45° C they were hardly detectable. A brief HS at 45° C (10 minutes), followed by incubation at 28° C, which also induced HSP synthesis, resulted in synthesis of this class of HSPs at levels up to 1.06 micrograms per 100 micrograms of total protein. Taking into consideration the previous data concerning the acquisition of thermotolerance in soybean seedlings, our estimation indicates that the accumulation of the 15- to 18-kilodalton HSPs to 0.76 to 0.98% of total protein correlated well with the establishment of thermotolerance. Of course, other HSPs, in addition to this group of proteins, may be required for the development of thermotolerance.     

Long-lived and short-lived heat-shock proteins in tobacco mesophyll protoplasts

We have studied modifications in the pattern of proteins synthesized by tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts when they are transferred from 25°C to 40°C. The synthesis of one group of proteins is practically unaffected by the heat shock. On the other hand, the synthesis of most other 25°C proteins is greatly reduced, while specific heat-shock proteins appear: 17 stable, neutral, major proteins, which are synthesized throughout the culture period at the higher temperature and which correspond to those observed in other organisms, and two basic proteins with a short lifetime and which are synthesized only during the first 2 hours of heat shock. We suggest that these latter proteins are regulatory peptides which intervene in the inhibition of 25°C syntheses.     

Cardiac Effects of Attenuating Gsα - Dependent Signaling

Aims

Inhibition of β-adrenergic signalling plays a key role in treatment of heart failure. Gsα is essential for β-adrenergic signal transduction. In order to reduce side-effects of beta-adrenergic inhibition diminishing β-adrenergic signalling in the heart at the level of Gsα is a promising option.

Methods and Results

We analyzed the influence of Gsα on regulation of myocardial function and development of cardiac hypertrophy, using a transgenic mouse model (C57BL6/J mice) overexpressing a dominant negative Gsα-mutant under control of the α-MHC-promotor. Cardiac phenotype was characterized in vivo and in vitro and under acute and chronic β-adrenergic stimulation. At rest, Gsα-DN-mice showed bradycardia (602 ± 13 vs. 660 ± 17 bpm, p<0.05) and decreased dp/dt_max (5037 ± 546- vs. 6835 ± 505 mmHg/s, p = 0.02). No significant differences were found regarding ejection fraction, heart weight and cardiomyocyte size. β-blockade by propranolol revealed no baseline differences of hemodynamic parameters between wildtype and Gsα-DN-mice. Acute adrenergic stimulation resulted in decreased β-adrenergic responsiveness in Gsα-DN-mice. Under chronic adrenergic stimulation, wildtype mice developed myocardial hypertrophy associated with increase of LV/BW-ratio by 23% (4.4 ± 0.2 vs. 3.5 ± 0.1 mg/g, p<0.01) and cardiac myocyte size by 24% (14927 ± 442 px vs. 12013 ± 583 px, p<0.001). In contrast, both parameters were unchanged in Gsα-DN-mice after chronic isoproterenol stimulation.

Conclusion

Overexpression of a dominant negative mutant of Gsα leads to decreased β-adrenergic responsiveness and is protective against isoproterenol-induced hypertrophy. Thus, Gsα-DN-mice provide novel insights into β-adrenergic signal transduction and its modulation in myocardial overload and failure.