Relationship between petal abscission and programmed cell death in Prunus yedoensis and Delphinium belladonna Depending on the species , the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid . Wilting at the end of petal life is due to programmed cell death -LRB- PCD -RRB- . It is not known whether the abscission of turgid petals is preceded by PCD . We studied some parameters that indicate PCD : chromatin condensation , a decrease in nuclear diameter , DNA fragmentation , and DNA content per nucleus , using Prunus yedoensis and Delphiniumbelladonna which both show abscission of turgid petals at the end of floral life . No DNA degradation , no chromatin condensation , and no change in nuclear volume was observed in P. yedoensis petals , prior to abscission . In abscising D.belladonna petals , in contrast , considerable DNA degradation was found , chromatin was condensed and the nuclear volume considerably reduced . Following abscission , the nuclear area in both species drastically increased , and the chromatin became unevenly distributed . Similar chromatin changes were observed after dehydration -LRB- 24 h at 60 °C -RRB- of petals severed at the time of flower opening , and in dehydrated petals of Ipomoea nil and Petunia hybrida , severed at the time of flower opening . In these flowers the petal life span is terminated by wilting rather than abscission . It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators , whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission . Dehydration of the petal cells , after abscission , was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration . This nuclear morphology has apparently not been described previously , for any organism . Introduction Floral life span depends on the fate of the petals . In several species the end of floral life span is due to petal withering or wilting . The petals in most of these species desiccate , and finally abscise or fall due to growth of the fruit . Only in a few species the desiccated petals remain attached to the fruit . In another large group of species the end of floral life span is determined by abscission of petals that are mostly or fully turgid . Wilting in petals , at the end of their lifespan , is due to a series of highly regulated processes that lead to cell death , thus is a form of programmed cell death -LRB- PCD ; van Doorn et al. 2003 -RRB- . Prior to visible PCD , many macromolecules in the petal cells are degraded to molecules that are suitable for phloem transport . These mobile molecules are transported to other parts of the plant -LRB- Winkenbach et al. 1970a , b -RRB- . Typical for PCD in wilting petals is the disappearance of most of the organelles and accumulation of degrading enzymes in the vacuole . This is followed by rupture of the vacuolar membrane , whereby several hydrolases are released . Tonoplast rupture in numerous cells results in their collapse , which is visible as wilting -LRB- Winkenbach 1970a , b ; van Doorn et al. 2003 -RRB- . PCD in several plant parts -LRB- Gunawardena et al. 2001 ; Wojciechowska and Olszewska 2003 -RRB- , including petals , is also usually accompanied by DNA degradation -LRB- van der Kop et al. 2003 ; Wagstaff et al. 2003 ; Yamada et al. 2006a , b -RRB- and chromatin condensation -LRB- Yamada et al. 2006a , b -RRB- . It is not known if the abscission of turgid petals is preceded by cellular changes that are indicative of PCD . We therefore investigated the time line of some of these changes , in Prunus yedoensis and Delphiniumbelladonna . In both species the petals abscise without any visible sign of turgor loss . We used DNA degradation -LRB- measured both on agarose gels and by using flow cytometry -RRB- , chromatin condensation , and a decrease in nuclear diameter as parameters that indicate PCD . According to our hypothesis these parameters would not detectably change , prior to petal abscission . Materials and methods Plant material Plants were grown in the garden of National Institute of Floricultural Science of Japan -LRB- P. yedoensis cv . Someiyoshino -RRB- or the greenhouse of the institute -LRB- D. belladonna cv . Bellamosum , Ipomoea nil cv . Violet , and Petunia hybrida cv . Double Duo Pink -RRB- . Just before the onset of flower opening , cut branches -LRB- P. yedoensis -RRB- or potted plants -LRB- the other species -RRB- were transferred into a growth chamber at 24 ± 1 °C , about 70 % relative humidity -LRB- RH -RRB- , and 12 h of daily illumination from cool-white fluorescent lights -LRB- 100 μmol m − 2 s − 1 -RRB- . The time to petal abscission and petal desiccation was observed daily , on plant material in the growth chamber . The timing of these symptoms was noted in six flowers from two plants . All determinations were made using petals that were collected from plant material in the chamber . DNA degradation DNA breakdown was detected by agarose gel analysis . Total DNA was extracted according to Yamada et al. -LRB- 2003 -RRB- . The amount of DNA was estimated by molecular absorption spectrophotometry . Equal amounts of total DNA -LRB- 3 μg -RRB- were immediately electrophoresed in a gel containing 3 % agarose and stained with SYBR Gold nucleic acid gel stain -LRB- Molecular Probes , Eugene , OR , USA -RRB- . The gel pattern was photographed , using an electronic UV transilluminator system -LRB- model FAS-III mini + DS-30 ; Toyobo , Tokyo , Japan -RRB- . The DNA degradation index was calculated according to Yamada et al. -LRB- 2006b -RRB- . Two biological replications , each containing several petals , were used for each stage of development . Depending on the species , two to several petals were used in each sample . Flow cytometry The petal was chopped in nuclear extraction buffer , part of the kit -LRB- High Resolution kit -RRB- for plant DNA analysis -LRB- Partec , Münster , Germany -RRB- , a reagent set provided by the manufacturer of the flow cytometer used -LRB- Ploidy Analyzer , Partec -RRB- . The extraction buffer in this kit was a low pH solution containing Triton X-100 . The extract was filtered through 50 μm nylon mesh . The medium with the isolated DNA masses -LRB- nuclei -RRB- was collected , the buffer with the fluorescent DNA stain 4 , 6-diamidino-2-phenylindole -LRB- DAPI -RRB- from a standard reagent set -LRB- Partec -RRB- was added , and the solution was vortex mixed . The DNA content of the isolated nuclei was analysed using the flow cytometer . The flow cytometer apparatus can be set to count all nuclei in a sample , and can be set to analyse a fixed number of nuclei . We here set it to detect 5,000 nuclei . DNA levels were obtained in a total of 5,000 nuclei . It should be noted that the large histogram peak was adjusted to 10 on the fluorescence scale . This means that nuclear condensation was not taken into account in the flow cytometry data . Two biological replications , each containing several petals , were used for each stage of development . Depending on the species , three to several petals were used in each sample . In each species , the number of petals was the same at all stages of development . Nuclear morphology ; chromatin condensation Nuclei were isolated from petals in the same way as for flow cytometry . Isolated nuclei were stained with DAPI , which stains DNA . The nuclei were observed under a fluorescence microscope -LRB- model PROVIS AX70 , Olympus , Tokyo , Japan -RRB- using U-excitation -LRB- 330 -- 385 nm -RRB- . Digital photography of nuclei was obtained for each stain using low light cool CCD camera -LRB- model DP30BW , Olympus -RRB- . Dehydration and ethylene treatment Flowers were excised when the petals were fully open . Cut flowers were placed in air in the growth chamber -LRB- 24 °C , about 70 % RH -RRB- or in a desiccator -LRB- at 60 °C -RRB- . For ethylene treatments , stems of cut flowers were placed in sterile distilled water , in a closed 70 l clear plastic box containing 2 μl/l ethylene . Petal abscission was checked at hourly intervals and abscised petals were collected immediately . The petals were then used instantly for the measurement of the various parameters mentioned . Statistics The number of replications is mentioned in the text and/or in the figure legends . Statistical analysis used one way analysis of variance , at P < 0.05 . All experiments were repeated at least once . Results Symptoms of petal life span cessation P. yedoensis flowers have five pale pink petals . After flower opening the petals remained attached to the flower for about 4 days , and then fell without visible wilting -LRB- Fig. 1a , P2 -RRB- . During the first day following abscission , petals that were held at 24 °C and 70 % RH changed colour from pale pink to darker pink , and subsequently became infiltrated with liquid -LRB- results not shown -RRB- . By the end of the first day following abscission , the petals started to shrivel at their margins -LRB- Fig. 1a , P3 -RRB- . By the end of the second day after abscission the petals had much more decreased in size -LRB- Fig. 1a , P4 -RRB- . The stages 2 -- 4 -LRB- pictures P2 -- P4 -RRB- in Fig. 1a use the same scale thus show the decrease in petal size . Fig. 1Petal abscission and petal dehydration in P. yedoensis , indicated by a P -LRB- a -RRB- and D.belladonna , indicated D -LRB- b -RRB- . Cut branches of -LRB- P. yedoensis -RRB- and potted plants -LRB- D.belladonna -RRB- stood in a growth chamber -LRB- 24 °C , about 70 % RH -RRB- . The time to abscission and to dehydration symptoms -LRB- in days from full flower opening -RRB- is shown in the left lower corner of the pictures -LRB- n = 6 -RRB- . Stages P1 and D1 : full flower opening ; P2 and D2 , petal abscission ; P3 and D3 , petals visibly dehydrated ; P4 and D4 , petals desiccated . All pictures have the same scale -LRB- bars = 10 mm -RRB- Flowers of Delphinium species contain five petals one of which has a large spur . The petals of D. belladonna fell about 7 days after flower opening -LRB- Fig. 1b , D2 -RRB- . They did not show any visible wilting symptom by the time of abscission . Two days after abscission the petals had slightly shrivelled -LRB- Fig. 1b , D3 -RRB- but by day-3 after abscission the shrivelling had become very clear -LRB- Fig. 1b , D4 -RRB- . The stages 2 -- 4 -LRB- pictures D2 -- D4 -RRB- in Fig. 1b have the same scale . DNA degradation Figure 2 shows the petal DNA agarose gels of one of two repeat experiments . The pixel ratios under the lanes are the means of two experiments . In both species , the agarose gels showed no DNA fragments at the time of flower opening -LRB- Fig. 2 , lanes P1 and D1 -RRB- . By the time of abscission , the petals of P. yedoensis did not show a statistically significant -LRB- P < 0.05 -RRB- increase in DNA fragmentation , compared with the time of floral opening -LRB- Fig. 2 , lane P2 -RRB- . In contrast , the petals of D. belladonna showed a considerable increase in DNA fragmentation by the time of abscission -LRB- Fig. 2b , lane D2 , pixel intensity had increased from 4.3 to 17.4 -RRB- . Following petal abscission the degree of DNA fragmentation , as observed on agarose gels , increased in P. yedoensis -LRB- Fig. 2a , compare lane P3 with lane P2 -RRB- and further increased in D. belladonna -LRB- Fig. 2b , compare lane D3 with lane D2 -RRB- . By the time of petal desiccation -LRB- P4 and D4 -RRB- the amount of fragmented DNA had become considerably lower than at the previous stage investigated , when the abscised petal showed visible wilting and dehydration -LRB- Fig. 2 -RRB- . Fig. 2Agarose gel analysis of total DNA isolated from the petals of P. yedoensis , annotated as P -LRB- a -RRB- and D. belladonna , annotated as D -LRB- b -RRB- . 3 μg of total DNA was extracted from the petals and electrophoresed in a 3 % agarose gel . Lanes P1 -- P4 and SD1 -- SD4 refer to DNA isolated from petals at stages P1 -- P4 and D1 -- D4 as shown in Fig. 1 . Stages P1 and D1 : full flower opening ; P2 and D2 , petal abscission ; P3 and D3 , petals visibly dehydrated ; P4 and D4 , petals desiccated . Pixel ratios below the lanes are means of two repeat experiments Nuclear morphology In P. yedoensis petals the nuclear morphology had not clearly changed by the time of petal abscission -LRB- Fig. 3a -RRB- . This is in contrast with D. belladonna where visual inspection showed that the nucleus was much smaller by the time of abscission , compared with the size observed at flower opening . The small nuclei of D. belladonna showed brighter DAPI fluorescence than the nuclei at flower opening . This indicates a high concentration of DNA per unit volume , thus chromatin condensation -LRB- Fig. 3b , D2 -RRB- . Nuclear morphology was studied using light microscopy , giving a two-dimensional impression only . For this reason nuclear size will be referred to as nuclear area . Fig. 3Morphology of DAPI-stained nuclei in the petals of P. yedoensis , indicated by a P -LRB- a -RRB- and D.belladonna , indicated D -LRB- b -RRB- . Two representative nuclei -LRB- -1 and -2 -RRB- are shown at each stage of development . Nuclei were isolated from the petals and observed using a fluorescence microscope under U-excitation . Bars : 5 μm -LRB- a -RRB- or 15 μm -LRB- b -RRB- . Stages as in Fig 1 . P1 and D1 1 : full flower opening ; P2 and D2 , petal abscission ; P3 and D3 , petals visibly dehydrated ; P4 and D4 , petals desiccated After abscission , during the period of dehydration , the nuclei of both species showed an increase in area -LRB- Fig. 3 -RRB- In P. yedoensis the nuclear area at stage P4 seemed even considerably larger than that at floral opening -LRB- Fig. 3a -RRB- . In D. belladonna the nuclear area was apparently similar at D4 compared to the stage of floral opening -LRB- D1 -RRB- , but it was ostensibly much larger compared with the area at the stage of petal abscission -LRB- P2 ; Fig. 3b -RRB- . In both species the nuclear periphery was no longer smooth at stage P4 or D4 -LRB- Fig. 3 -RRB- . In both species investigated , numerous nuclei showed the morphology shown in Fig. 3 , stages P4 and D4 . The area of the remaining nuclei did not increase as much , whilst a few nuclei had not changed in nuclear area with respect to controls at flower opening . Flow cytometry of nuclei at various stages prior to and after abscission Flow cytometry of nuclei stained for DNA -LRB- with DAPI -RRB- shows the distribution of nuclei in fluorescence classes . The X-axis in Fig. 4 is divided in numerous classes of increasing fluorescence , and the Y-axis shows the number of nuclei per fluorescence class . Flow cytometry of 5.000 petal nuclei at the time of P. yedoensis flower opening showed a peak of nuclei at phase G0/G1 -LRB- which was set at 101 on the arbitrary logarithmic scale of the X-axis -RRB- , and no mitotic nuclei -LRB- G2/M -RRB- , which have a fluorescence about two times that of normal G0/G1 nuclei . By the time of petal abscission , no change had occurred in the DNA fluorescence histogram -LRB- Fig. 4a , P2 -RRB- . Two days after abscission , the height of the G0/G1 peak had diminished but the peak had not shifted . This is shown by mixing nuclei from day-0 to -6 -LRB- Fig. 4a , P1 + P4 -RRB- . The data suggest that a small amount of DNA had become degraded by day-2 after abscission . Fig. 4Fluorescence of DAPI-stained nuclei . Histograms were obtained by flow cytometry of 5,000 nuclei isolated from the petals of P. yedoensis -LRB- indicated by a P -RRB- as shown in a , and petals of D.belladonna -LRB- D -RRB- shown in b. Petals were severed from cut twigs placed in water -LRB- Prunus -RRB- or potted plants -LRB- Delphinium -RRB- , which stood both in a growth chamber -LRB- 24 °C , about 70 % RH -RRB- . Stages as in Fig. 1 . P1 and D1 : full flower opening ; P2 and D2 , petal abscission ; P3 and D3 , petals visibly dehydrated ; P4 and D4 , petals desiccated By the time of petal abscission in D. belladonna , flow cytometry revealed a decrease in the height of the G0/G1 phase nuclei , and several nuclei with a fluorescence less than that of G0/G1 phase nuclei -LRB- Fig. 4b , D2 -RRB- . During the 3 days following abscission the G0/G1 peak height further decreased and the number of nuclei with fluorescence lower than G0/G1 further increased . No clear shift in the G0/G1 peak was observed : a mix of nuclei at day-0 and -10 did not result in a bimodal curve -LRB- Fig. 4b , D1 + D4 -RRB- . The results indicate extensive DNA degradation , before , during , and after abscission . Effects of ethylene Ethylene treatment greatly hastened the time to petal abscission , in both species -LRB- Fig. 5a , compared with Fig. 1a -RRB- . In both species , the rate of petal dehydration after abscission was not much affected by the ethylene treatment -LRB- Fig. 5a -RRB- compared with the control without ethylene -LRB- Fig. 1a -RRB- . For example , 3 days after petal fall in D. belladonna the petals had extensively shrivelled , independent of the time of petal fall -LRB- day-7 in controls and day-2 in ethylene-treated flowers -RRB- . Fig. 5Effect of ethylene on petal abscission and DNA fluorescence of nuclei in the petals of P. yedoensis and D.belladonna . Time to petal abscission and petal dehydration symptoms -LRB- a -RRB- and flow cytometric determination of 5,000 DAPI-stained nuclei -LRB- b -RRB- of ethylene-treated branches of P. yedoensis and potted plants of D.belladonna . Ethylene treatment is annotated by E. Compare stages of development with untreated controls shown in Fig. 1 , and with fluorescence data of controls in Fig. 4 . The left lower corner of the pictures series a show the times to abscission -LRB- EP2 and ED2 -RRB- and the time to dehydration symptoms -LRB- both expressed in days from full flower opening ; n = 6 -RRB- . Stages P1 and D1 : full flower opening ; EP2 and ED2 , petal abscission ; EP3 and ED3 , onset of petal dehydration ; EP4 and ED4 , petals desiccated . All pictures have the same scale -LRB- bars = 10 mm -RRB- In both species , flow cytometry revealed no effect of ethylene treatment on DNA fluorescence at the time of petal fall , compared to the fluorescence at the time of flower opening -LRB- Fig. 5b -RRB- . After petal fall , a clear shift to lower DNA fluorescence of the G0/G1 peak of fluorescence was observed in P. yedoensis -LRB- Fig. 5b , upper sequence -RRB- , which was not found in controls without ethylene treatment -LRB- Fig. 4 , upper sequence -RRB- . A small shift to lower DNA fluorescence -LRB- Fig. 5b , lower sequence -RRB- , also not found in controls -LRB- Fig. 4 lower sequence -RRB- , was also observed after ethylene treatment of D. belladonna . Superimposition of the stages P1 and EP4 -LRB- control and ethylene-treated stage 4 of P. yedoensis -RRB- showed two peaks of DNA fluorescence -LRB- Fig. 5b , upper sequence -RRB- . A similar tendency was observed by superimposing the stages D1 and ED3 -LRB- Fig. 5b , lower sequence -RRB- . Dehydration of petals cut at flower opening Flowers were harvested just after opening and subjected to a dehydration treatment at 60 °C for 24 h . The treatment resulted in a petal FW of about 20 % of that of controls . Preliminary experiments showed that such a treatment resulted in a decrease of FW , and in changes of other parameters such as DNA degradation and nuclear morphology , that were very similar to dehydration on the laboratory bench -LRB- about 20 °C and 70 % RH -RRB- for about 96 h. Treatment of P. yedoensis flowers at 60 °C for 24 h -LRB- Fig. 6a , DP -RRB- resulted in a slight increase in petal DNA degradation , as shown on agarose gel -LRB- Fig. 6b , DP -RRB- . In contrast , this treatment induced a large increase in DNA degradation in D. belladonna petals -LRB- Fig. 6b , DD -RRB- . Fig. 6Effect of dehydration on DNA degradation , DNA fluorescence of nuclei , and nuclear morphology in the petals of P. yedoensis -LRB- P -RRB- and D.belladonna -LRB- D -RRB- . Dehydration in the two species is indicated as DP and DD , respectively . a Flower morphology before -LRB- P1 and D1 -RRB- and after -LRB- DP and DD -RRB- dehydration at 60 °C for 24 h. Photographs show small branches with fully opened flowers and desiccated flowers of P. yedoensis -LRB- P1 and DP -RRB- and the same for individual flowers of D.belladonna -LRB- D1 and DD -RRB- . All pictures have the same scale -LRB- bars = 10 mm -RRB- . b Agarose gel analysis of total DNA isolated from the petals . c Flow cytometric determination of nuclei isolated from the petals . d Morphology of nuclei in the petals . Two representative nuclei -LRB- -1 and -2 -RRB- are shown for each species , after the dehydration treatment -LRB- DP and DD -RRB- . Bars = 5 μm -LRB- P1 and DP -RRB- or 15 μm -LRB- D1 and DD -RRB- The dehydration treatment also had a small effect on DNA fluorescence of nuclei from P. yedoensis petals -LRB- Fig. 6c -RRB- . Superimposition of the nuclei prior to and after treatment showed no shift to lower DNA fluorescence -LRB- Fig. 6c , P1 + DP -RRB- . In D. belladonna , the dehydration treatment resulted in lower G0/G1 DNA fluorescence , and many nuclei with a lower than G0/G1 fluorescence -LRB- Fig. 6c -RRB- . Superposition of the treatment and the control showed no shift of the G0/G1 peak of DNA fluorescence -LRB- Fig. 6c , D1 + DD -RRB- . Following the dehydration treatment , the nuclei of P. yedoensis had apparently somewhat increased in area , but no clear increase in nuclear area was discernable in D. belladonna -LRB- Fig. 6d -RRB- . The chromatin distribution in the nuclei , after the dehydration treatment -LRB- Fig. 6d -RRB- was similar to that after dehydration due to abscission -LRB- Fig. 3 -RRB- . Visual inspection indicated that about 80 % of all nuclei had the morphology shown in Fig. 6c , d . The area of the remaining nuclei had not as much increased . Dehydration of petals in species that show petal wilting -LRB- Ipomoea and Petunia -RRB- Similar dehydration treatments -LRB- 60 °C for 24 h -RRB- were carried out with I. nil and Petunia hybrida , species in which the petals wilt by the end of flower life span . Wilting is shown as inrolling in I. nil , which occurs at about 12 h after flower opening , in plants held at 24 °C -LRB- Fig. 7a -RRB- . Wilting is observed after 7 days at 24 °C in flowers attached to P. hybrida plants -LRB- Fig. 7a -RRB- . Fig. 7Effect of dehydration on fresh weight , DNA degradation , and DNA fluorescence of nuclei in the petals of I. nil and P. hybrida . Potted plants were grown in a greenhouse and then placed in a climate-controlled chamber -LRB- 24 °C , about 70 % RH -RRB- . Petals were detached from flowers that had just opened , and were placed at 60 °C for 24 h to dehydrate . In a the time until about half of each petal had wilted -LRB- in days from full flower opening ; n = 6 -RRB- is shown in the left lower corner of the pictures . a Morphology of the flowers , at opening -LRB- left -RRB- and at petal wilting -LRB- right -RRB- . All pictures have the same scale -LRB- bars = 10 mm -RRB- . b Agarose gel analysis of total DNA isolated from the petals . c , d Flow cytometric determination of 5,000 DAPI-stained nuclei isolated from the petals of I. nil -LRB- c -RRB- and P. hybrida -LRB- d -RRB- . Ip , Ipomoea ; Pe , Petunia , D , dehydration Flowers were detached from potted plants , just after full opening . Fig. 7b shows considerable DNA fragmentation on agarose gels , after the dehydration treatment -LRB- 60 °C for 24 h -RRB- , in both species tested . Flow cytometry of Ipomoea petals held at 60 °C for 24 h showed a large decrease in DNA fluorescence , compared to controls of Ipomoea -LRB- Ip -RRB- before desiccation -LRB- Fig. 7c , compare DIp with Ip , whereby D means dehydrated -RRB- . Fig. 7d shows that DNA degradation was also found in Petunia -LRB- Pe -RRB- . Superimposition of the fluorescence in controls and the treated samples showed two peaks -LRB- Fig. 7c , d -RRB- . The morphology of DAPI-stained nuclei of I. nil and P. hybrida , after petal desiccation at 60 °C for 24 h , is shown in Fig. 8 . Compared to nuclei of petals in flowers that had just fully opened -LRB- two panels upper left -RRB- , relatively large parts not stained with DAPI were observed -LRB- two panels upper right -RRB- . In I. nil the nuclear area had not become clearly increased , compared to those in petals that had not been desiccated -LRB- Fig. 8a -RRB- , but inflated nuclei were observed in P. hybrida -LRB- Fig. 8b -RRB- . Visual inspection indicated that about 80 % of all nuclei showed the morphology shown in Fig. 8b . The area of the remaining nuclei had not as much increased . Fig. 8Effect of dehydration on nuclear morphology in the petals of I. nil and P. hybrida . Nuclei were isolated from the petals of I. nil -LRB- a -RRB- and P. hybrida -LRB- b -RRB- after the indicated treatments , then observed using a fluorescence microscope under U-excitation . For comparison , the nuclei are shown during PCD of petals that remained attached to the plant -LRB- for data of Ipomoea and Petunia see also Yamada et al. 2006a , b -RRB- . Stage S1 : full flower opening ; S2 : onset of petal wilting ; S3 : full petal wilting ; and S4 : petals desiccated but still attached to the flower . Bars = 10 -LRB- μm Both in I. nil -LRB- Fig. 8a , S1 -- S4 -RRB- and in P. hybrida -LRB- Fig. 8b , S1 -- S4 -RRB- the nuclear morphology after the dehydration treatment was quite different from the one observed during normal PCD . During PCD of petals that remained attached to the plant the area of the nuclei became increasingly smaller , whereas the brightness of DNA fluorescence remained high at least until the nuclei had become quite small . Discussion In reports on animal cells the suggestion that PCD occurs is usually backed up by showing changes such as phosphatidylserine externalization to the outer leaflet of the plasma membrane , caspase activation , and DNA degradation . Only the addition of a number of such parameters seems indicative of PCD . In the present study we took massive DNA degradation -LRB- measured in two ways -RRB- , chromatin condensation , and a decrease in nuclear diameter as indicators of PCD . In other studies on petal PCD , it was shown that these parameters were each correlated with other indicators of PCD and also correlated with visible PCD symptoms such as petal wilting -LRB- Yamada et al. 2003 , 2006a , b -RRB- . We therefore suggest that these parameters , which separately represent only circumstantial evidence for PCD , are , when occurring together , adequate to indicate PCD . We hypothesized that PCD parameters will not show in petal cells , prior to the abscission of turgid petals . The results of D. belladonna petals contradict the hypothesis , while those of P. yedoensis are in agreement with it . The data suggest that the degree of PCD , prior to abscission of turgid petals , depends on the species . The present data indicate that abscission of fully turgid petals may coincide with an advanced stage of PCD in many petal cells . The absence of visible wilting in abscising petals , therefore , does not seem to show absence of PCD . This is so because the wilting that is often associated with PCD is quite a late stage in the PCD process . In petals the timing of PCD and that of abscission may be regulated independently . Independent regulation would explain the large variation of their relative timing , among the Angiosperms . In many species PCD comes much earlier than abscission -LRB- species showing petal wilting at the end of floral life -RRB- , whereas in other species abscission comes first . In most species in which floral life span is ended by petal wilting , the petals eventually abscise , even when most of the petal surface has become desiccated . In these species the petal base , where abscission takes place , remains alive . Only in a few species fully desiccated petals are torn-off by the growing fruit , or -LRB- in still other species -RRB- remain attached to the fruit -LRB- van Doorn and Stead 1997 ; van Doorn 2001 -RRB- . The agarose gel data -LRB- Fig. 2 -RRB- showed substantial DNA degradation prior to and during abscission in D. belladonna and less DNA degradation prior to and during petal fall in P. yedoensis . Flow cytometry data -LRB- Fig. 4 -RRB- confirmed the agarose gel data . DNA degradation seems a relatively late PCD process . Mass protein degradation , a late process , may serve as a yardstick . In Ipomoea petals , bulk degradation of proteins and DNA occurred at about the same time -LRB- Winkenbach 1970a , b -RRB- . In Iris petals , 40 % of total DNA had become degraded -LRB- van der Kop et al. 2003 -RRB- by the time of the onset of mass protein degradation -LRB- Pak and van Doorn 2005 -RRB- . In Alstroemeria petals DNA breakdown -LRB- Wagstaff et al. 2003 -RRB- started slightly before the decrease in protein content -LRB- Wagstaff et al. 2002 -RRB- . When using agarose gels to show DNA breakdown during PCD , a ladder of DNA fragments is often observed , at least in association with apoptosis in animal cells . DNA laddering on gels is the result of oligonucleosomal cleavage , resulting in DNA sequences of multiples of about 180 bp . In contrast to apoptosis in animal cells , the DNA breakdown pattern during plant PCD is quite variable and depends on the system . For example , during petal PCD clear DNA laddering without a background of DNA fragments of various lengths is rarely found . Most reports show DNA laddering against a background of fragments of various lengths -LRB- Orzáez and Granell 1997 ; Wagstaff et al. 2003 ; Yamada et al. 2003 -RRB- or only fragments of various lengths -LRB- van der Kop et al. 2003 -RRB- . The present results fall in the last category . Nuclear morphology at the time of petal abscission also suggested advanced PCD in the petal cells of D. belladonna and little PCD in those of P. yedoensis . The nuclei of D. belladonna were much smaller and showed bright DAPI fluorescence , indicating chromatin condensation . No chromatin condensation was observed in P. yedoensis petals , but here it might possibly have occurred between the stages P2 and P3 and therefore may have been missed . We found that most but not all nuclei underwent the changes shown at the stages 2 -- 4 of Fig. 3 . The difference might relate to differences in PCD progress , within the petal . It is known that cells associated with the vascular bundle undergo PCD at a later stage than the other petal cells . In some species the mesophyll cells also show earlier PCD than the epidermis cells -LRB- van Doorn et al. 2003 -RRB- . Little is known about the physiology of petal cells , after abscission . The cells of abscised petals , as long as they remain alive , will be subject to a considerable decrease in water potential . Using young detached corollas -LRB- petals -RRB- of Ipomoea flowers , we observed that FW was about 65 % of initial after 24 h on the laboratory bench -LRB- at about 20 °C and 70 % RH -RRB- and 50 % of initial after 48 h -LRB- unpublished data -RRB- . This indicated that some physiological processes would occur at least during day-1 and -2 , but that these processes would be influenced by lower water potential . Both agarose gel -LRB- Fig. 2 , P3 and D3 -RRB- and flow cytometry analysis -LRB- Fig. 4 -RRB- showed DNA degradation after the petals had abscised . The data indicate that the PCD process , once started , goes on even under conditions of increasing water stress . However , by the time of total desiccation of the petals , the amount of degraded DNA on gels was smaller than at the time of visible petal wilting of the abscised petals -LRB- Fig. 2 , P4 and D4 -RRB- . This indicated that the DNA breakdown became halted by that time . This could be due to the fact that there was no more DNA , or might relate to the very low water potential in the tissue . After petal abscission , the petal nuclei unexpectedly increased in size . In both species the chromatin became unevenly distributed . The chromatin became also much less condensed , as indicated by decreasing DAPI brightness . The increase in nuclear size and uneven and reduced DAPI brightness were also observed after dehydration of young D. belladonna and P. yedoensis petals . In these experiments flowers were cut from the plant just after floral opening and then subjected to a desiccation treatment -LRB- 24 h at 60 °C -RRB- . This treatment , apart from inducing dehydration , also involved an increase in temperature . The effects did not seem to be due to high temperature though , as preliminary experiments had shown that 72 -- 96 h desiccation at room temperature had the same effects as 24 h treatment at 60 °C . This indicates that the effect was due to dehydration rather than high temperature , and that it was not only taking place in cells that were relatively old , but also in much younger cells . I. nil and P. hybrida flowers both exhibit petal wilting rather than abscission at the end of floral life span . An increase in nuclear volume and a decrease in DAPI brightness were also found in the nuclei , if the flowers of these species were severed when just fully open and then subjection to a 24 h 60 °C desiccation treatment . Preliminary experiments with these species had also shown that 72 -- 96 h desiccation at room temperature had the same effects as 24 h treatment at 60 °C . These results indicate that the particular nuclear morphology observed after dehydration in P. yedoensis and D. belladonna is also found in the petals of other species , which show petal wilting rather than petal abscission at the end of flower life . The particular nuclear morphology , therefore , seemed related to the low water potential as a result of the dehydration treatment . After the dehydration treatment in I. nil or P. hybrida the nuclear morphology was quite different from the one observed during wilting as the final phase of PCD . At the time of the normal senescence-related petal wilting in Ipomoea -LRB- Yamada et al. 2006b -RRB- and Petunia -LRB- Yamada et al. 2006a -RRB- flowers , the nuclei had become much smaller than at previous phases of development , and had a rather bright DAPI fluorescence . The desiccation treatment , therefore , induced changes quite different from those observed during PCD : a large nucleus with faint DAPI fluorescence rather than a small one with bright fluorescence . It is concluded that the tested parameters indicated advanced stages of PCD prior to petal abscission in D. belladonna whereas PCD was much less obvious by the time of petal abscission in P. yedoensis . Dehydration of the petal cells , after abscission , was associated with a remarkable change in nuclear morphology and chromatin distribution . Similar changes in the nucleus were found after dehydration of younger petal cells , and after dehydration of the petal cells in two other species . This nuclear morphology has apparently not been described previously .

O O O B O B I I O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O B O O O O O O O O O B I I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

abscission
dna degradation
flow cytometry
programmed cell death
wilting


Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation Elevated cardiac troponin-I -LRB- cTnI -RRB- levels have been demonstrated in serum of patients without acute coronary syndromes , potentially via a stretch-related process . We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins . Cultured cardiomyocytes were treated with -LRB- 1 -RRB- Gly -- Arg -- Gly -- Asp -- Ser -LRB- GRGDS , n = 22 -RRB- to stimulate integrins , -LRB- 2 -RRB- Ser -- Asp -- Gly -- Arg -- Gly -LRB- SDGRG , n = 8 -RRB- that does not stimulate integrins , or -LRB- 3 -RRB- phosphate-buffered saline -LRB- control , n = 38 -RRB- . Cells and media were analyzed for intact cTnI , cTnI degradation products , and matrix metalloproteinase -LRB- MMP -RRB- -2 . Cell viability was examined by assay of lactate dehydrogenase -LRB- LDH -RRB- activity and by nuclear staining with propidium iodide . GRGDS-induced integrin stimulation caused increased release of intact cTnI -LRB- 9.6 ± 3.0 % -RRB- as compared to SDGRG-treated cardiomyocytes -LRB- 4.5 ± 0.8 % , p < 0.001 -RRB- and control -LRB- 3.0 ± 3.4 % , p < 0.001 -RRB- . LDH release from GRGDS-treated cardiomyocytes -LRB- 15.9 ± 3.8 % -RRB- equalled that from controls -LRB- 15.2 ± 2.3 % , p = n.s. -RRB- , indicating that the GRGDS-induced release of cTnI is not due to cell necrosis . This result was confirmed by nuclear staining with propidium iodide . Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls -LRB- both p < 0.05 -RRB- . However , despite the ability of active MMP2 to degrade cTnI in vitro , integrin stimulation in cardiomyocytes was not associated with cTnI degradation . The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins . Introduction Troponins are myofibrillar proteins involved in the regulation of actin -- myosin interaction , thereby controlling contraction and relaxation . Cardiac troponins -LRB- cTnT , cTnT , and cTnC -RRB- are predominantly bound , via tropomyosin , to actin filaments of sarcomeres , with only a small proportion of cTnT -LRB- 6 -- 8 % -RRB- and cTnI -LRB- 3 -- 8 % -RRB- found in the soluble cytoplasmic pool -LSB- 1 -RSB- . Due to high cardiac specificity , serum concentrations of cTnI and cTnT are well-established diagnostic and prognostic markers of irreversible myocardial damage in acute coronary syndromes -LSB- 9 -RSB- . However , elevated serum levels of cardiac troponins have also been observed in patients without acute coronary syndromes in whom irreversible myocardial cell injury was not a prominent aspect -LSB- 8 , 11 , 20 -RSB- . Several studies reported elevated serum levels of troponins in patients with cardiomyopathy -LSB- 29 -RSB- , heart failure -LSB- 18 -RSB- , unstable angina pectoris -LSB- 14 -RSB- , pulmonary embolism -LSB- 17 -RSB- , renal insufficiency -LSB- 5 -RSB- , and in ultra-endurance athletes -LSB- 19 -RSB- . The exact mechanism underlying the release of troponins in the absence of necrosis , without lethal disruption of the sarcolemma , remains to be elucidated and most likely differs from the release of troponins due to necrotic cell death . A potential explanation for the cTnI release without lethal sarcolemmal disruption is the cellular release of proteolytic cTnI degradation products . Indeed , in isolated rat hearts , Feng et al. -LSB- 6 -RSB- showed degradation of cTnI upon increasing preload , which was independent of ischemia . They proposed that the cTnI degradation might be caused by increased myocardial stretch per se . Mechanical stretch of cardiomyocytes , as occurs during pressure or volume overload , initiates a cascade of intracellular signals , including increased intracellular calcium concentration , increased intracellular NO formation , and the activation of intracellular proteases such as MMP2 and MMP14 -LSB- 28 , 36 -RSB- . MMP2 is able to degrade cTnI intracellularly -LSB- 39 -RSB- and may be involved in the stretch-induced release of cTnI and its degradation products . In contrast , other studies suggest that cTnI leaks from reversibly damaged cardiomyocytes as an intact non-degraded molecule -LSB- 8 , 20 -RSB- . Mechanical stretch of cardiomyocytes is sensed by stimulation of integrins , which are transmembrane glycoprotein receptors that link the extracellular matrix -LRB- ECM -RRB- to the intracellular cytoskeleton -LSB- 26 -RSB- . Integrins act as mechanotransducers and bidirectional signaling molecules and participate in overload-induced hypertrophic and post-infarct remodeling -LSB- 26 -RSB- . The Arg -- Gly -- Asp -LRB- RGD -RRB- sequence that is present in fibronectin and other ECM proteins is an agonist for integrins -LSB- 27 -RSB- , which can be used to simulate myocardial mechanical stretch in vitro . We hypothesize that the stretch-related process through which cTnI can be released from viable cardiomyocytes is mediated by integrin stimulation . The purpose of the present study is therefore to investigate whether -LRB- 1 -RRB- RGD-induced integrin stimulation causes release of intact and/or fragmented cTnI from cardiomyocytes in the absence of necrotic cell death , -LRB- 2 -RRB- integrin stimulation is associated with MMP2 activation and MMP2-related cTnI degradation , and -LRB- 3 -RRB- cTnI release in the absence of necrosis differs from the release of cTnI in the presence of necrosis . Materials and methods Primary cultures of neonatal cardiomyocytes Primary cultures of cardiomyocytes were prepared from the ventricles of 2-day-old Wistar rats as described previously -LSB- 24 -RSB- . Briefly , ventricles were minced , and cells were isolated enzymatically using collagenase type I -LRB- CLS , Lakewood , NJ , USA -RRB- at 37 °C . The cell suspension was centrifuged , and the cell pellet was resuspended in growth medium containing Ham 's F10 -LRB- Flow Laboratories , Irvine , UK -RRB- , 10 % fetal bovine serum , 10 % horse serum -LRB- HS -RRB- , 100 U/ml penicillin , and 100 μg/ml streptomycin -LRB- all from Invitrogen , Breda , The Netherlands -RRB- . The cells were seeded in 6-cm diameter Primaria ® - coated plastic culture dishes -LRB- Falcon , Becton Dickinson , Etten-Leur , The Netherlands -RRB- , and after 45 min , non-adherent cells representing the cardiomyocytes were collected and plated in six-wells plates -LRB- ∅ 35 mm per well , Costar , Corning , NY , USA -RRB- at a density of ≈ 1.5 × 106 cells per well . Cardiomyocytes were incubated in a humidified incubator at 37 °C and 5 % CO2 and culture medium -LSB- 1:1 -LRB- v/v -RRB- mixture of DMEM -LRB- Invitrogen -RRB- and Ham 's F10 supplemented with 5 % HS , penicillin -LRB- 100 U/ml -RRB- and streptomycin -LRB- 100 μg/ml -RRB- -RSB- was refreshed after 20 h and 48 h . After 3 days of culturing , a monolayer of spontaneously beating cardiomyocytes had formed . The investigations had the approval of the Animal Experiments Committee of the LUMC according to Dutch law . Experimental protocol for integrin stimulation Three days after cell isolation , cardiomyocyte cultures were washed twice in HEPES-buffered salt solution -LRB- HBSS -RRB- , containing -LRB- in millimole per liter -RRB- NaCl -LRB- 125 -RRB- , KCl -LRB- 5 -RRB- , MgSO4 -LRB- 1 -RRB- , KH2PO4 -LRB- 1 -RRB- , CaCl2 -LRB- 2.5 -RRB- , NaHCO3 -LRB- 10 -RRB- , d-glucose -LRB- 5 -RRB- , HEPES -LRB- 20 -RRB- , pH 7.4 at 37 °C . Cardiomyocytes were incubated for 24 h in 1 ml of HBSS supplemented with -LRB- 1 -RRB- the pentapeptide Gly -- Arg -- Gly -- Asp -- Ser containing the GRGDS sequence -LRB- Sigma , St. Louis , MO , USA -RRB- at various concentrations -LRB- 100 , 200 , or 300 μg/ml -RRB- to stimulate integrins , with -LRB- 2 -RRB- Ser -- Asp -- Gly -- Arg -- Gly -LRB- SDGRG , the reverse sequence of GRGDS , Sigma -RRB- , which does not affect integrin function , at a concentration of 300 μg/ml , or with -LRB- 3 -RRB- phosphate-buffered saline -LRB- PBS -RRB- as a control . After 24 h , medium was separated from the cells , cells were taken up in 1 ml of ice-cold lysis buffer -LRB- 100 mmol/l Tris -- HCl , 0.1 % Tween , pH 7.5 -RRB- , both cell and medium samples were kept on ice awaiting assay of lactate dehydrogenase -LRB- LDH -RRB- activity on the same day , and subsequently stored at − 20 °C for later cTnI and MMP2 assays . Troponin-I ELISA Purified human heart cTnI -LRB- Calbiochem , LaJolla , CA , USA -RRB- was dissolved in urea/Tris buffer according to the manufacturer 's instructions . Monoclonal mouse anti-cTnI clone 19C7 -LRB- directed against amino acid sequence 40 -- 50 -RRB- and monoclonal mouse anti-cTnI clone 6F9 -LRB- directed against amino acid sequence 189-195 -RRB- were purchased from HyTest -LRB- Turku , Finland -RRB- . The enzyme-linked immunosorbent assay -LRB- ELISA -RRB- was based on the sandwich principle as described previously -LSB- 13 -RSB- . The percentage cTnI released from each culture equalled . Detection limit of the assay was 6 ng/ml . Inter-assay variability was 6 and 10 % at a TnI concentration of 200 and 25 ng/ml , respectively . The intra-assay variability was 6 % at TnI concentrations of 100 ng/ml . Western blot analysis Protein extracts were size-fractionated on NuPage Novex 12 % Bis -- Tris gels -LRB- Invitrogen -RRB- and transferred to Hybond PVDF membranes -LRB- Amersham Biosciences , Roosendaal , The Netherlands -RRB- . Non-specific binding sites were blocked using blocking solution consisting of 20 g/l ECL advance blocking agent -LRB- Amersham Biosciences -RRB- in TBS-Tween -LRB- 10 mmol/l Tris -- HCl , 150 mmol/l NaCl , 0.05 % Tween pH 8.0 -RRB- . Membranes were incubated for 1 h with the anti-TnI antibody -LRB- K83085G , Biodesign , Saco , ME , USA -RRB- , which detects various cTnI degradation products . After four washes in TBS-Tween , membranes were incubated with horseradish peroxidase-labeled secondary antibody -LRB- rabbit anti-goat IgG , Santa Cruz Biotechnology , Heidelberg , Germany -RRB- . Chemiluminescence was induced by ECL advance detection reagent -LRB- Amersham Biosciences -RRB- and detected by exposure to Hyperfilm ECL -LRB- Amersham Biosciences -RRB- . LDH activity assay Necrotic cell death was quantified by assay of LDH activity released from cells into medium according to Wroblewski and Ladue -LSB- 41 -RSB- . LDH activities of medium and cell samples were determined using a spectrophotometer -LRB- Ultrospec 3000 , Pharmacia Biotech , Roosendaal , The Netherlands -RRB- at 25 °C . The total LDH activity in each culture , i.e. , cell + medium , was determined , and the LDH activity in cell and medium samples was expressed as a percentage of total LDH activity . The detection limit of the LDH assay was 5 U/l , the intra-assay variability was 3 % -LRB- 110 U/l -RRB- and 6 % -LRB- 48 U/l -RRB- , and inter-assay variability was 12 % -LRB- 110 U/l -RRB- . Nuclear staining with propidium iodide An additional assessment of cell death was performed by quantifying the nuclei of necrotic cells using propidium iodide and fluorescence microscopy . For this purpose , cardiomyocyte cultures were treated with PBS , GRGDS , and SDGRG for 24 h , then incubated with propidium iodide -LRB- 5 mg/l , Molecular Probes , Eugene , OR , USA -RRB- for 15 min in the dark , washed in PBS , and viewed under a fluorescence microscope -LRB- Zeiss , Hamburg , Germany -RRB- at 535/617-nm excitation/emission . Subsequently , cultures were incubated with digitonine -LRB- 10 μmol/l -RRB- for 5 min to induce necrotic cell death in all cardiomyocytes . The extent of necrosis equalled the number of necrotic nuclei before digitonine incubation as a fraction of the number of necrotic nuclei after digitonine incubation -LRB- 100 % -RRB- . In vitro degradation of purified cTnI by active MMP2 To study whether active MMP2 is able to degrade cTnI in vitro , purified human cardiac TnI -LRB- 500 pg/μl , Calbiochem -RRB- was incubated with human recombinant active MMP2 -LRB- 1.5 ng/μl , Oncogene , San Diego , CA , USA -RRB- in enzyme buffer at 37 °C for 0 , 60 , and 120 min . In a separate series of experiments , MMP2 was preincubated with an inhibitor of MMP2 , o-phenanthroline -LRB- 100 μmol/l -RRB- for 15 min . As a control , cTnI was incubated without active MMP2 at 37 °C . Intact cTnI and cTnI degradation products were visualized by Western blotting . Zymography Gelatinolytic activities of MMP2 in cell and medium samples of control , GRGDS - and SDGRG-treated cardiomyocytes were analyzed by zymography as described previously -LSB- 12 , 33 -RSB- . Briefly , cell samples -LRB- tenfold concentrated by freeze-drying for 18 h -RRB- and medium samples -LRB- unconcentrated -RRB- were prepared in sample buffer and were loaded on gels . After electrophoresis , the gels were washed twice in 2.5 % Triton X-100 , were washed in enzyme buffer , and were incubated in enzyme buffer at 37 °C overnight . Gels were stained with amino black and subsequently destained . Gelatinase activity was quantified by measuring the extent of gelatin digestion using a scanning densitometer -LRB- 2202 Ultrascan , LKB , Paramus , NJ , USA -RRB- . Experimental protocol for necrotic cell death Three days after cell isolation , cardiomyocyte cultures were washed twice in HBSS , pH 7.4 , at 37 °C , and subsequently incubated in 1 ml of HBSS , supplemented with sodium azide -LRB- 1 mmol/l , n = 7 -RRB- at 37 °C . After 24 h of incubation , medium was separated from the cells , cells were taken up in 1 ml of ice-cold lysis buffer , both cell and medium samples were kept on ice awaiting assay of lactate dehydrogenase -LRB- LDH -RRB- activity on the same day , and subsequently stored at − 20 °C for later cTnI assay . Statistics Results are expressed as mean ± SD . Statistical analysis was performed by Student 's t test and by one-way analysis of variance followed by Bonferroni 's post hoc test . Differences were regarded as statistically significant if p < 0.05 . SPSS14 for Windows -LRB- SPSS , Chicago , IL , USA -RRB- was used for statistical analysis . Results Effect of integrin stimulation on release of cTnI Cell and medium samples were assayed for intact cTnI by ELISA -LRB- Fig. 1a -RRB- and for cTnI degradation products using Western blotting -LRB- Fig. 2a -- d -RRB- . Cellular cTnI content in ten cultures was 0.27 ± 0.10 μg per culture -LRB- 100 % -RRB- . Control cardiomyocytes had released ≈ 3 % of total cTnI , probably as a result of cell death of a few cardiomyocytes during 24 h of incubation . GRGDS treatment at a concentration of 100 or 200 μg/ml did not show considerable release of cTnI -LRB- ≈ 2 % -RRB- . However , a concentration of 300 μg/ml GRGDS resulted in a significant extra release of intact cTnI -LRB- to ≈ 10 % -RRB- , compared with control cardiomyocytes -LRB- p < 0.001 -RRB- and cardiomyocytes treated with 100 or 200 μg/ml GRGDS -LRB- both p < 0.001 ; Fig. 1a -RRB- . Incubation of cardiomyocytes with 300 μg/ml SDGRG , the reverse sequence of GRGDS that has no effect on integrin function , did not induce a significant extra release of intact cTnI -LRB- Fig. 1a -RRB- . In addition , Fig. 2b and d shows that cTnI was released as an intact protein of 29 kDa and that integrin stimulation is not associated with the release of cTnI degradation products . Fig. 1a Release of cardiac troponin-I -LRB- cTnI -RRB- from cardiomyocytes incubated with PBS -LRB- control , n = 38 -RRB- , GRGDS -LRB- 100 μg/ml , n = 5 ; 200 μg/ml , n = 4 ; 300 μg/ml , n = 22 -RRB- , or SDGRG -LRB- 300 μg/ml , n = 8 -RRB- , detected by ELISA . b Release of LDH from cardiomyocytes incubated with PBS -LRB- control , n = 38 -RRB- , GRGDS -LRB- 300 μg/ml , n = 22 -RRB- , or SDGRG -LRB- 300 μg/ml , n = 8 -RRB- . -LRB- * p < 0.001 vs control , # p < 0.001 vs GRGDS 100 , $ p < 0.001 vs GRGDS 200 , ‡ p < 0.001 vs GRGDS 300 , and § p < 0.001 vs SDGRG 300 -RRB- Fig. 2Western blots of intact cTnI -LRB- 29 kDa -RRB- and cTnI degradation products present in cell samples -LRB- left , 2.5 × diluted -RRB- and medium samples -LRB- right , 10 × concentrated -RRB- of cardiomyocytes incubated with PBS -LRB- control -RRB- , GRGDS -LRB- 300 μg/ml , a and b -RRB- , SDGRG -LRB- 300 μg/ml , c and d -RRB- , or sodium azide -LRB- 1 mmol/l , e and f -RRB- for 24 h Effect of integrin stimulation on cell viability Necrotic cell death was quantified by the release of LDH activity from cardiomyocytes into the medium -LRB- Fig. 1b -RRB- . Cellular LDH activity of ten cultures was 0.47 ± 0.24 U per culture -LRB- 100 % -RRB- . In control cardiomyocytes , ≈ 15 % of total LDH was released in 24 h , which confirms previous findings of cellular necrosis in cardiomyocytes after 24 h in serum-free medium -LSB- 25 -RSB- . LDH release from integrin-stimulated cardiomyocytes -LRB- GRGDS , 300 μg/ml -RRB- equalled LDH release from control cardiomyocytes -LRB- Fig. 1b -RRB- . Surprisingly , cardiomyocytes treated with SDGRG -LRB- 300 μg/ml -RRB- for 24 h showed significantly less LDH release -LRB- 9.5 % -RRB- than control and GRGDS-treated cardiomyocytes -LRB- both p < 0.001 -RRB- . Assessment of cell death using nuclear staining with propidium iodide showed a similar pattern . The percentage of necrotic cells in controls -LRB- 7.2 ± 2.1 % , n = 8 -RRB- equalled that of GRGDS-treated cardiomyocytes -LRB- 8.5 ± 2.2 % , n = 8 , p = N.S. -RRB- . Again , the percentage of necrotic cells in SDGRG-treated cardiomyocytes was significantly lower -LRB- 6.2 ± 1.9 % , n = 8 -RRB- compared with GRGDS-treated cardiomyocytes -LRB- p < 0.05 -RRB- , confirming the results of the LDH assay . These results demonstrate that integrin stimulation by GRGDS participates in the release of cTnI from cardiomyocytes in the absence of necrosis . Effect of integrin stimulation on MMP2 activity In control experiments , we verified that recombinant active MMP2 is able to degrade purified cTnI in vitro in a number of fragments of 26 , 14 and 8 kDa -LRB- Fig. 3 -RRB- . However , as we observed no cTnI degradation upon integrin stimulation -LRB- Fig. 2 -RRB- , we studied whether this was due to the lack of MMP2 activation during integrin stimulation , measuring the zymographic gelatinolytic activities of MMP2 . In control cardiomyocytes , gelatinolytic MMP2 activities were detected in both cell and medium samples , indicating a baseline level of MMP2 in cultured cardiomyocytes -LRB- Fig. 4 -RRB- . Total MMP2 levels were higher in medium than in cells -LRB- ≈ 90 vs ≈ 10 % of total MMP2 , respectively -RRB- . Fig. 3Western blot of intact cTnI -LRB- 29 kDa -RRB- and cTnI degradation products after in vitro incubation of purified intact cTnI without and with active MMP2 and with active MMP2 in combination with a MMP2 inhibitor -LRB- o-phenanthroline -RRB- for 0 , 60 , and 120 minFig . 4Gelatinolytic activities of MMP2 isoforms in cell samples -LRB- left , 10 × concentrated -RRB- and medium samples -LRB- right , unconcentrated -RRB- of cardiomyocytes incubated with PBS -LRB- control , n = 12 -RRB- , GRGDS -LRB- 300 μg/ml , n ≥ 9 -RRB- , or SDGRG -LRB- 300 μg/ml , n ≥ 3 -RRB- for 24 h In cells samples , several gelatinolytic activities of MMP2 were detected corresponding to a rodent-specific glycosylated MMP2 -LRB- 75 kDa -RRB- , proMMP2 -LRB- 72 kDa , the major gelatinase activity -RRB- , an active intermediate MMP2 -LRB- 64 kDa -RRB- , and active MMP2 -LRB- 62 kDa ; Fig. 4 , left -RRB- . GRGDS-treated cardiomyocytes demonstrated significantly higher levels of cellular proMMP2 -LRB- 72 kDa -RRB- and active MMP2 -LRB- 64 + 62 kDa -RRB- than control and SDGRG-treated cardiomyocytes -LRB- Fig. 4 , left -RRB- , whereas levels of glycosylated MMP2 -LRB- 75 kDa -RRB- were unaffected by GRGDS . In medium samples -LRB- Fig. 4 , right -RRB- , several gelatinolytic activities of MMP2 were detected , corresponding to a rodent-specific glycosylated MMP2 -LRB- 75 kDa -RRB- , proMMP2 -LRB- 72 kDa -RRB- , and active MMP2 -LRB- 62 kDa -RRB- , respectively . GRGDS-induced integrin stimulation resulted in significantly higher medium levels of glycosylated MMP2 -LRB- 75 kDa -RRB- and active MMP2 -LRB- 62 kDa -RRB- compared to medium samples of control and SDGRG-treated cardiomyocytes -LRB- both p < 0.05 ; Fig. 4 , right -RRB- . Medium levels of proMMP2 -LRB- 72 kDa -RRB- were unaffected by integrin stimulation . Release of cTnI from necrotic cardiomyocytes To compare the release of cTnI from viable cardiomyocytes during GRGDS-induced integrin stimulation with the release of cTnI upon necrotic cell death , cardiomyocyte cultures were treated with sodium azide -LRB- 1 mmol/l -RRB- to induce necrotic cell death . After 24 h of azide-treatment , LDH release from cardiomyocytes was 73.4 ± 10.5 % -LRB- n = 7 -RRB- , indicating extensive necrotic cell death . Release of intact cTnI from these necrotic cardiomyocytes was 32.7 ± 13.8 % -LRB- n = 7 -RRB- . Notably , in contrast to the release of intact cTnI from integrin-stimulated cardiomyocytes -LRB- Fig. 2b -RRB- , cardiomyocyte necrosis leads to the release of at least four cTnI degradation products with molecular weights of 26 , 20 , 17 , and 12 kDa -LRB- Fig. 2f -RRB- . Discussion The major findings of this study are that integrin stimulation in cardiomyocytes by GRGDS leads to -LRB- 1 -RRB- release of intact cTnI from viable cardiomyocytes in the absence of necrosis and -LRB- 2 -RRB- increased levels of intracellular and extracellular MMP2 activity that , however , does not result in degradation of cTnI into its fragments . This study therefore indicates that the release mechanism of cTnI from viable cardiomyocytes upon integrin stimulation differs from cTnI release from necrotic cardiomyocytes , which is associated with extensive cTnI degradation . Elevated plasma cTnI levels are frequently found in pathological conditions in which irreversible myocardial cell injury is not a prominent aspect . In patients with heart failure , elevated plasma cTnI levels have been reported with normal plasma levels of CK-MB -LSB- 2 , 18 -RSB- . Some studies have suggested that elevated serum levels of cTnI may be the result of myocardial strain -LSB- 15 , 20 -RSB- , as occurs during pressure or volume overload . Logeart et al. -LSB- 15 -RSB- studied 71 patients with heart failure and found in 19 patients elevated cTnI concentrations associated with LV remodeling and increased plasma brain natriuretic peptide levels . They postulated that increased LV wall strain may have led to the increased cTnI release , but necrotic cell death was not excluded in that study . In acute pulmonary embolism , several reports showed elevated cTnI levels -LSB- 17 , 21 -RSB- possibly related to the presence of myocardial strain . In addition , Feng et al. -LSB- 6 -RSB- demonstrated that increased preload in isolated rat hearts was sufficient to cause release of cTnI as the result of myocardial stretch independent of ischemia . Overload-induced stretch at the cardiomyocyte level is sensed by integrins , mechanotransducers molecules that link the extracellular matrix to the intracellular cytoskeleton -LSB- 32 -RSB- . In the present study , we showed that GRGDS-induced integrin stimulation of cardiomyocytes resulted in a threefold increased release of cTnI that occurred in the absence of necrosis , as cell viability remained unchanged by GRGDS treatment compared with control cardiomyocytes . Control cardiomyocytes demonstrated baseline levels of necrosis after 24 h , consistent with previous findings of cell death in cardiomyocytes after 24 h in serum-free medium -LSB- 25 -RSB- . Surprisingly , we found significantly less necrosis in SDGRG-treated cardiomyocytes than in control cardiomyocytes with both LDH assay and nuclear staining with propidium iodide . The mechanism responsible for this protective effect is unlikely to be related to integrin function but may be related to protection against necrosis exerted by certain proteins such as serum components . Several studies have suggested that release of cTnI from viable cardiomyocytes , without lethal disruptions of the cardiomyocyte sarcolemma , does not involve intact cTnI -LRB- 29 kDa -RRB- but rather proteolytic degradation products of cTnI formed by intracellular proteases as MMP2 and calpain-I -LSB- 6 , 7 -RSB- . In the present study , we demonstrated that purified intact cTnI can indeed be degraded by active MMP2 in vitro . MMPs are a family of zinc-containing enzymes involved in degradation of the extracellular matrix during tissue remodeling -LSB- 34 -RSB- . However , MMP2 also acts intracellularly and may , therefore , be responsible for degradation of myofilament proteins including cTnI and myosin light chain-1 -LRB- MLC-1 -RRB- -LSB- 30 , 39 -RSB- . MMPs are synthesized by a variety of cells , including cardiomyocytes -LSB- 4 -RSB- , in a latent form -LRB- proMMP -RRB- that is activated by either proteolytic cleavage or by conformational changes induced by cytokines , reactive oxygen species , and peroxynitrite -LSB- 22 , 35 , 38 -RSB- . Previously , Wang et al. -LSB- 37 -RSB- have demonstrated that mechanical stretch of neonatal cardiomyocytes induced expression and activation of MMP2 . In addition , integrin stimulation by GRGDS has been reported to upregulate MMP2 expression in fibroblasts and certain tumor cell lines -LSB- 31 , 40 -RSB- , suggesting an important role for integrins in regulating MMP2 activity . In the present study , we showed that GRGDS-induced integrin stimulation of cultured cardiomyocytes was indeed associated with increased activity of intracellular proMMP2 and active MMP2 and increased release of active MMP2 into the medium . In cardiomyocytes incubated with SDGRG , neither cellular levels nor medium levels of proMMP2 or active MMP2 were altered compared to control cardiomyocytes , indicating that increased MMP2 activity is specific for GRGDS-induced integrin stimulation . However , despite the increase of MMP2 activity during integrin stimulation and the ability of active MMP2 to degrade purified cTnI in vitro , integrin stimulation was not associated with cTnI degradation . If cTnI degradation had occurred , we would have been able to detect that in this system -LRB- Figs. 3 and 2f -RRB- . The reason for the lack of cTnI degradation in the presence of GRGDS-induced MMP2 activation is unclear . The discrepancy between the in vitro degradation of purified cTnI by active MMP2 and the lack of cTnI degradation in GRGDS-treated cardiomyocytes may be explained by a difference in specific MMP2 activity . The MMP2 activity per nanogram cTnI in the study with purified cTnI was fourfold higher than that in cardiomyocytes treated with RGD . In addition , purified cTnI may be more susceptible to MMP2 cleavage than cTnI that is complexed to cTnT , cTnC , and tropomyosin . In cardiomyocytes , 93 -- 97 % of cTnI is complexed to cTnT , cTnC , and tropomyosin which may protect the MMP2 cleavage sites on the cTnI molecule . The mechanism responsible for the release of intact cTnI from viable integrin stimulated cardiomyocytes may be leakage of free intact cTnI from the cytosolic pool . Several studies have demonstrated that mechanically induced transient disruptions -LRB- wounding -RRB- of the sarcolemma are a constitutive event in vivo -LSB- 3 , 10 , 16 , 23 -RSB- . This mechanism is responsible for the release of proteins , such as myocyte-derived growth factors -LRB- fibroblast growth factors 1 and 2 -RRB- , which are released despite the lack of a classic signal peptide sequence normally associated with exocytotic secretion . These mechanically induced alterations in cardiomyocyte 's sarcolemmal permeability may similarly be involved in the release of cTnI from the cytosolic pool of cardiomyocytes in the absence of necrotic cell death . Further study is needed to address this issue . Conclusions The present study demonstrates that viable cardiomyocytes release cTnI as an intact protein by a stretch-related mechanism mediated by integrins . This finding may possibly explain why , in several pathological conditions , plasma cTnI levels are elevated in the absence of myocardial necrosis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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