despite advances in treatment , cardiovascular disease is still the leading cause of morbidity and mortality in the western world .
a prolonged cardiac hypertrophic state leads to heart failure and is commonly accompanied by complex changes in protein / peptide levels in plasma and/or in the heart .
chromogranin a ( human : chga ; rodent : chga ) , a 48- to 52-kda acidic secretory protein in homeothermic vertebrates ( helle 2004 ; montero - hadjadje et al .
2003 ; winkler and fischer - colbrie 1992 ) , is a proprotein giving rise to several peptides of biological importance such as the dysglycemic hormone pancreastatin ( pst ) ( gayen et al . 2009 ;
1986 ) , vasodilator and cardiosuppressive vasostatin ( vst ) ( aardal et al . 1993 ; tota et al . 2008 ) , antiadrenergic , antihypertensive and cardioinhibitory peptide catestatin ( cst ) ( angelone et al . 2008 ; mahapatra et al . 2005 ; mahata et al .
1997 , 2000 , 2010 ) and we14 which acts as an autoantigen in type 1 diabetes ( stadinski et al .
although initially described in the chromaffin granules of the adrenal medulla , subsequent studies demonstrate widespread distribution of chga / chga in endocrine tissues ( fischer - colbrie et al .
1985 ; oconnor 1983 ) and in the nervous system ( mahata et al . 1991
winkler s group elegantly demonstrated the presence of chga and chromogranin b ( chgb ) in the secretory granules in atrial myoendocrine cells of rat heart and their co - localization with atrial natriuretic peptide ( anp ) ( steiner et al .
subsequently , in rat , chga has been detected in purkinje conduction fibers , in both atrium and ventricle , as well as in h9c2 cardiomyocytes ( weiergraber et al .
a recent confocal microscopical study clearly shows co - localization of chga with b - type natriuretic peptide ( bnp ) in human ventricular myocytes of dilated and hypertrophic hearts ( pieroni et al .
the plasma concentration of chga is elevated in established human essential hypertension ( oconnor et al . 2008 ; takiyyuddin et al . 1995 ) and in rodent model of genetic hypertension ( oconnor et al . 1999 ) .
serum chga levels show distinct correlation with severity of cardiac dysfunction and act as a predictive factor for mortality in patients with chronic heart failure ( ceconi et al .
recent studies demonstrate a close and independent association between serum chga levels and outcome in patients with acute coronary syndromes ( estensen et al .
2006 ; jansson et al . 2009 ; omland et al . 2003 ) and in patients with acute ( dieplinger et al .
elisa assays with four different monoclonal antibodies show a significant amount of chga only in pathologic myocardium ( > 0.05  g / g of tissue ) ( pieroni et al . 2007 ) .
the circulating levels of cst decrease in patients with essential hypertension ( oconnor et al .
2002 ) and targeted ablation of the chga gene in mice increases blood pressure , which can be  rescued  by replacement with cst ( mahapatra et al .
we have shown that in addition to its hypotensive action in rodents ( kennedy et al .
2005 ) and in humans ( fung et al . ) , cst exerts cardiosuppressive effects on the isolated langendorff - perfused rat heart under both basal and chemically stimulated conditions ( angelone et al .
thus , in addition to its important role in the control of blood pressure , cst is emerging as a peptide that has direct cardiovascular actions , suggesting that the negative inotropism and lusitropism of cst may be important components of its hypotensive action .
in addition , we have recently found that plasma chga is elevated , and its processing to cst is diminished , in hypertension ( oconnor et al .
therefore , in the present study , we sought to determine the presence of chromogranin / secretogranin proteins in murine heart and the processing of chga to cst by using biochemical and mass spectrometry techniques .
our data reveal the presence of chga , chgb and scg2 and several chga - derived peptides containing cst motif in murine heart .
tissue extraction very young ( 10  days , 1-month old ) , young adult ( 2-month , 3-month - old ) and adult ( 6-month - old ) mice with mixed genetic background ( 129svjxc57bl/6 ) were used in this study .
mice were kept in a 12-h dark / light cycle and were fed standard chow diet .
animal care and sacrifice were carried out according to the guidelines of institutional animal care and utilization committee .
heart and adrenal glands were collected from isoflurance - anesthetized mice and were freshly frozen in liquid nitrogen .
tissues were homogenized using a tissuemizer in tris - maleate buffer ( 10  mm tris - maleate , ph  7.0 , sucrose 0.2  m , edta 2  mm , protease inhibitors cocktail and phosphatase inhibitors cocktail ; sigma , st .
louis , mo , usa ) , centrifuged at 8,000 g for 30  min and the supernatants were collected . cardiomyocyte culture and protein extraction pups ( 34  days old ) were sacrificed ; heart tissues were collected in ads buffer ( hepes sodium salt 20  mm , nacl 116  mm , d - glucose 5.5  mm , kcl 5.4  mm , na2hpo4 9  mm , mgso4 0.4  mm ) and digested with collagenase type ii ( 0.1  mg/100  ml of ads buffer ; worthington biochemical ) .
cells were pre - plated for 1  h in uncoated plates to allow the fibroblasts to attach .
the non - adhered cells were then plated in gelatin - coated plates and cultured in dmem - low glucose with 10% fbs and pen / strep for 48  h. after that , medium was replaced by dmem with 1% fbs and the cells were cultured for additional 24  h. spontaneously beating confluent monolayers were established 2448  h after plating . at 72  h , cells were washed with pbs twice , scrapped in lysis buffer ( 20  mm tris - cl , ph  7.4 , edta 1  mm , nacl 75  mm , triton x-100 0.5% , bme 0.1% v / v ) .
cells were then centrifuged at 14,000 g for 10  min and the supernatants were collected .
protein estimation protein concentration was determined using bio - rad protein assay reagent ( bio - rad laboratories , hercules , ca , usa ) .
sds - page and immunoblot proteins were separated using a 10% sds - page or a 1020% tricine gel ( novex precast gel ; invitrogen , san diego , ca , usa ) and processed for western blot analysis as described before ( biswas et al .
we used rabbit polyclonal anti - human cst ( 1:3,000 ) , goat polyclonal anti human chgb ( 1:500 , sc-1489 ; santa cruz biotechnology , santa cruz , ca , usa ) , rabbit polyclonal ani - human scg2 ( 1:1,000 ) , and goat polyclonal anti - actin ( 1:500 , sc-1615 ; santa cruz biotechnology ) for immunoblot studies .
immunofluorescence cardiomyocyte cultures were grown on coverslips for 72  h , fixed with 2.5% paraformaldehyde and processed for immunocytochemistry as described before ( biswas et al .
the primary antibodies used were : rabbit polyclonal anti - human chga ( 1:1,000 dilution ) , goat anti - chgb ( 1:100 dilution , sc-1489 ) , rabbit anti - human scg2 ( 1:2,000 dilution ) , goat anti - beta myosin heavy chain ( myh , 1:100 , sc-12117 ) and goat anti - atrial natriuretic peptide ( anp , 1:100 , sc-18811 ) .
purification of cst - containing peptides frozen heart tissue ( 6-month - old animals ) was extracted in 1  m acetic acid , boiled for 5  min , and homogenized .
the supernatant ( 1  mg protein ) was subjected to reverse phase hplc purification ( symmetry c18 column , 5  m , 4.6    250  mm ; waters , milford , ma , ysa ) .
proteins were eluted with a gradient ( 2560% over 1585  min ) of acetonotrile at a flow rate of 0.5  ml / min .
fractions ( 1  min ) were collected , lyophilized and spotted on a nitrocellulose membrane , and immunostained with a cst - specific antibody .
the cst - positive fractions from different run were lyophilized , pooled and subjected to mass spec analysis by electrospray method .
anti - cst immunoprecipitation frozen heart tissues ( 1-month - old animals ) were homogenized in tris - maleate buffer and the resulting supernatants were subjected to immunoprecipitation .
tissue extracts ( 2  mg and 1  mg ) were pre - cleared by incubating with normal rabbit serum in 1 immunoprecipitation buffer ( ip buffer : 10  mm tris - cl , ph  7.5 , triton x-100 0.1% , nacl 140  mm ) .
antibody complexes were pulled out using protein g plus / protein a agarose ( calbiochem , san diego , ca , usa ) and washed 2 times with 1x ip buffer .
the bound proteins were eluted from the beads either in tricine loading dye for immunoblot analysis or with acetonitrile / water / tfa for mass spec analysis .
mass spec analysis electrospray and maldi tof analysis were carried out on a qtof ( qstar elite ) and voyager destr mass spectrometer respectively at the biomolecular and proteomics mass spectrometry facility , ucsd , department of chemistry and biochemistry as described before .
the resulting peptide masses were analyzed in the program protein prospector ( http://prospector.ucsf.edu ) to identify the possible fragments and were listed .
cst eia cst eia was carried out using a cst specific eia kit ( ek-053 - 29 ) from phoenix pharmaceutical ( burlingame , ca , usa ) statistical analysis biochemical data are expressed as the meansem .
multiple comparisons were made using one - way anova followed by bonferroni s post hoc test .
to determine the expression of chromogranin / secretogranin proteins , primary culture of cardiomyocytes from 3- to 4-day - old pups were analyzed by immunocytochemistry .
deconvolution microscopy shows punctuate staining of chromogranin / secretogranin proteins , which indicates discrete vesicular distribution of chga ( fig . 
1c ) proteins in the cytoplasm of murine cardiomyocytes . as a positive control , we detected vesicular staining for the myocyte specific protein ( myh : 60% of cells ) ( fig . 
1d ) and atrial natriuretic peptide ( anp : 4% of cells ) ( fig .  1e ) .
cardiomyocyte cell extracts showed several distinct low molecular mass ( ranging from 25 to 37  kda ) cst - positive fragments , along with low abundance full - length chga ( ran at 75  kda ) ( fig . 
the localization of chga ( a ) , chgb ( b ) and scg2 ( c ) were assessed in primary culture of cardiomyocytes , derived from 3- to 4-day - old pups .
cardiomyocytes were stained with primary antibodies specific for each chromogranin / secretogranin proteins and the cardiomyocyte specific markers such as myosin heavy chain ( myh ) ( d ) and atrial natriuretic peptide ( anp ) ( e ) .
the secondary antibodies used were donkey anti - rabbit igg - alex flur 488 ( green fluorescence , 1:250 ) and donkey anti - goat igg - alex flur 594 ( red fluorescence , 1:350 ) .
cells were examined by deconvolution microscopy using a 60 objective ( scale bar 5  m ) for the upper panel and a 20 objective ( scale bar 10  m ) for the lower panel .
f cardiomyocyte cell extracts ( lane 1 20  g protein and lane 2 40  g protein ) were analyzed by sds - page followed by immunoblot with a cst specific antibody .
g freshly frozen adult ( 6-month - old ) mice heart and adrenal tissues were homogenized in tris - maleate buffer and were subjected to sds - page and immunoblot analysis .
heart extracted proteins ( 75  g ) were run by sds - page for the detection of chga ( by rabbit anti - human cst ) , chgb ( by goat anti - human chgb ) and scg2 ( by rabbit anti - human scg2 ) .
adrenal extracts ( proteins in lane 1 2  g , lane 2 15  g , lane 3 30  g , and lane 4 60  g ) were also run by sds - page for the detection of chga ( by rabbit anti - human cst ) .
for adrenal extract the full - length chga signal was shown in the upper panel and the low molecular mass fragments were shown in the lower panel the expression of chromogranin / secretogranin proteins in murine heart .
the localization of chga ( a ) , chgb ( b ) and scg2 ( c ) were assessed in primary culture of cardiomyocytes , derived from 3- to 4-day - old pups .
cardiomyocytes were stained with primary antibodies specific for each chromogranin / secretogranin proteins and the cardiomyocyte specific markers such as myosin heavy chain ( myh ) ( d ) and atrial natriuretic peptide ( anp ) ( e ) .
the secondary antibodies used were donkey anti - rabbit igg - alex flur 488 ( green fluorescence , 1:250 ) and donkey anti - goat igg - alex flur 594 ( red fluorescence , 1:350 ) .
cells were examined by deconvolution microscopy using a 60 objective ( scale bar 5  m ) for the upper panel and a 20 objective ( scale bar 10  m ) for the lower panel .
f cardiomyocyte cell extracts ( lane 1 20  g protein and lane 2 40  g protein ) were analyzed by sds - page followed by immunoblot with a cst specific antibody .
g freshly frozen adult ( 6-month - old ) mice heart and adrenal tissues were homogenized in tris - maleate buffer and were subjected to sds - page and immunoblot analysis .
heart extracted proteins ( 75  g ) were run by sds - page for the detection of chga ( by rabbit anti - human cst ) , chgb ( by goat anti - human chgb ) and scg2 ( by rabbit anti - human scg2 ) .
adrenal extracts ( proteins in lane 1 2  g , lane 2 15  g , lane 3 30  g , and lane 4 60  g ) were also run by sds - page for the detection of chga ( by rabbit anti - human cst ) . for adrenal extract the full - length chga signal
was shown in the upper panel and the low molecular mass fragments were shown in the lower panel to identify cst - containing fragments , we carried out sds - page followed by cst immunoblot in heart and adrenal extracts .
adult heart extracts revealed three cst - positive fragments with masses of 37 , 31 and 15  kda ( fig . 
murine heart also showed faint immunoreactivity at 75  kda , which is the apparent migratory behavior of full - length chga in sds - page gel .
in addition , we detected a very high molecular mass form of chga , possibly representing the proteoglycan form . because of the presence of a high number of acidic amino acid residues within the primary structure of chromogranin / secretogranin proteins , these proteins run anomalously on sds - page and their apparent molecular weight differs from the weight deduced by primary amino acid sequence .
in contrast to chga , chgb showed a major full - length form and also some low molecular mass fragments ( fig . 
scg2 , on the other hand , showed extensive aggregation or modification as evidenced by the presence of high molecular mass protein as major signal along with some low molecular mass fragments .
compared to the heart extract , the full - length chga appeared to be highly prevalent in the adrenal extract ; the major cst positive fragment ran at 75  kda .
after exposing the lower part of the membrane for a longer period of time , we detected comparable processing products of chga between adrenal gland and heart as evidenced by the presence of smaller , processed forms of chga with masses of 37 , 27 , and 15  kda in the adrenal extract ( fig . 
although both the adrenal gland and heart express the processed forms , the ratio of processed to full - length form is much higher in the heart compared to the adrenal gland indicating an extensive processing of chga in the heart as compared to the adrenal gland .
although chga is overexpressed in humans with essential hypertension and in genetic model of rodent hypertension , cst showed diminished expression in hypertensive subjects and in normotensive individuals with a family history positive for hypertension .
since hypertension is a late penetrating disease , and cst plays crucial roles in the development of hypertension , we sought to determine whether processing of chga to cst in murine heart is altered with age .
the full - length chga signal ( ran 75  kda ) as well as the intensity of the 37  kda chga c - terminal fragment were increased in older animals ( fig . 
however , the intensity of a low molecular mass cst - specific peptide ( less than 15  kda ) decreased in the heart of older animals ( fig . 
consistent with immunoblot findings , cst eia displayed a significant reduction of endogenous cst peptide in the heart extract of older mice ( 3090  days old ) as compared to younger ( 10-day - old ) mice ( fig . 
heart from different age groups of mice ( age in days : 10 , 30 , 60 and 90 ) were collected , homogenized and subjected to sds - page and immunoblot using a cst - specific antibody ( rabbit anti - human cst ) ( a ) .
b the endogenous cst concentrations were measured from tissue extracts using a cst eia kit .
cst concentrations are expressed in pg/g of protein age - dependent processing of chga to cst in murine heart .
heart from different age groups of mice ( age in days : 10 , 30 , 60 and 90 ) were collected , homogenized and subjected to sds - page and immunoblot using a cst - specific antibody ( rabbit anti - human cst ) ( a ) .
b the endogenous cst concentrations were measured from tissue extracts using a cst eia kit .
cst concentrations are expressed in pg/g of protein mouse heart extract was purified using the rp - hplc column ( fig .  3 ) and the fractions collected for slot blot analysis .
from the anti - cst slot blot , two fractions eluting at 48% ( 6163  min ) and 54% ( 7376  min ) acetonitrile were positive for cst immunoreactivity ( fig . 
3b ) . to evaluate the apparent molecular mass of chga c - terminal cst - containing fragments in these fractions , each fraction was lyophilized , pooled and subjected to mass spec analysis .
electrospray mass spec analysis revealed the presence of several cst - spanning peptides in heart extracts ( table  1 ) . among them , the peptides of mass 5,051.60 and 5,641.11 , representing chga356398 and chga357404 or chga358405 , respectively , had considerable overlap with the n - terminal part of the long cst sequence , mchga356384 .
the peptides of mass 4,757.50 , 5,244.85 and 2,478.34 contained a significant part of the n - terminal of short cst sequence , mchga364384 ( table  1 ) . 

mice hearts ( 6 months old ) were homogenized in acetic acid and subjected to reverse phase hplc purification .
the proteins were eluted ( the chromatogram showing protein elution as measured at 214  nm with time is shown in ( a ) ) , with a gradient of acetonitrile and the fractions were collected , lyophilized and spotted onto nitrocellulose membrane to probe with a cst - specific antibody ( b ) .
fraction numbers are shown on the right of the respective slot blot ( b)table  1catestatin - related peptides detected by mass spectrometry from reverse phase hplc purification of heart extract .
the cst - immuno positive fractions from reverse phase hplc , as detected by slot blot , were lyophilized , and subjected to electrospray mass spectrometry .
the resulting peptides generated in the catestatin region are listed in boldmass ( monoisotopic ) submittedmass ( monoisotopic ) matchedsequencecleavage sites4,989.674,989.37325364e / er / s4,989.54349390k / ep / s5,148.745,148.52328369e / es / f5,057.515,057.52339380k / rp / g3,061.553,061.50342367s / rk / l4,341.314,341.17344380m / dp / g3,034.663,034.56346371q / lr / t4,989.784,989.54349390k / ep / s5,051.65,051.47356398r / le / a5,641.115,643.69357404/358405l / e or e / ge / e or e / g3,294.723,294.67360387e / dg / w4,757.54,751.41364403d / rf / e5,244.855,244.63365408r / se / e2,478.342,478.36367387m / kg / w1,754.941,754.87369383l / sq / l2,676.452,676.41369390/370391s / f or l / ss / s or p / s5,187.645,187.51369412l / ss / a2,369.222,369.2376395y / gd / s3,828.983,828.88378409f / re / e2,008.992,009.00379395r
/ dd / smouse catestatin sequence:[kr]legeddpdrsmklsfrtraygfrdpgpql[rr]long cst : mchga356384short cst : mchga364384 identification of cst - related peptides from heart extract .
mice hearts ( 6 months old ) were homogenized in acetic acid and subjected to reverse phase hplc purification .
the proteins were eluted ( the chromatogram showing protein elution as measured at 214  nm with time is shown in ( a ) ) , with a gradient of acetonitrile and the fractions were collected , lyophilized and spotted onto nitrocellulose membrane to probe with a cst - specific antibody ( b ) .
fraction numbers are shown on the right of the respective slot blot ( b ) catestatin - related peptides detected by mass spectrometry from reverse phase hplc purification of heart extract .
the cst - immuno positive fractions from reverse phase hplc , as detected by slot blot , were lyophilized , and subjected to electrospray mass spectrometry .
the resulting peptides generated in the catestatin region are listed in bold mouse catestatin sequence : [ kr]legeddpdrsmklsfrtraygfrdpgpql[rr ] long cst : mchga356384 short cst : mchga364384 since young animals showed more low molecular mass cst fragments ( < 15  kda ; see fig . 
2a ) , we sought to purify cst related peptides from 1-month - old mice with an alternate strategy of immunoprecipitation .
sds - page analysis ( on a 1020% tricine gel ) of immunoprecipitated complex showed several fragments ranging from 20 to 150  kda ( fig . 
4a , left panel ) . besides these high molecular mass fragments , the longer exposure of the lower part of the membrane revealed the presence of a peptide that ran at 7  kda ( fig .  4a , middle panel ) .
as a positive control , we ran synthetic human cst ( hchga352372 ; molecular mass    2.4  kda ) ( fig . 
although we did not achieve a good separation between 2 and 7  kda protein even on a 1020% gel , our results indicate the presence of a low molecular mass ( in the range of 27  kda ) fragment in the anti - cst immunoprecipitated complex .
the maldi - tof analysis showed a major peptide of 11  kda corresponding to the c - terminal chga derived fragment chga283384 ( fig . 
several peptides with cst motif have been identified . among them , peptides of mass 4,097 and 6,224 overlapped with the n - terminal sequence of short cst , mchga364384 ( table  2 ) . 

mice heart ( 1-month - old animal ) were homogenized in tris - maleate buffer and subjected to immunoprecipitation from 2 to 1  mg protein with anti - cst antibody .
the antigen - antibody complexes were pulled down with protein a / g beads and subjected to immunoblot analysis ( lane 1 ip from 2  mg protein , lane 2 ip from 1  mg protein ) ( a ) . in ( a ) , rabbit anti - human cst antibody was used to detect cst - related peptides .
the middle panel of ( a ) showed the darker exposure of the lower part of the gel .
the right panel of ( a ) showed the running position of the synthetic human cst in the same gel .
for maldi - tof ( b ) , proteins eluted from the beads with acetonitrile / water / tfa were lyophilized and analyzed by mass spectable  2catestatin spanning peptides detected by mass spectrometry from immunoprecipitation of heart extract .
proteins were eluted from the beads with acetonitrile / water / tfa , lyophilized and analyzed by maldi - tof .
the detected peptides are summarized belowmass ( average ) submittedmass ( average ) matchedsequence4,0974,097.65339372/3403734,098.623653994,2284,225.823383725,7335,736.463313775,738.533423895,9615,961.673333816,2246,218.8636541711,92011,920.02283384 anti - cst immunoprecipitation from the heart extract .
mice heart ( 1-month - old animal ) were homogenized in tris - maleate buffer and subjected to immunoprecipitation from 2 to 1  mg protein with anti - cst antibody .
the antigen - antibody complexes were pulled down with protein a / g beads and subjected to immunoblot analysis ( lane 1 ip from 2  mg protein , lane 2 ip from 1  mg protein ) ( a ) . in ( a ) , rabbit anti - human cst antibody was used to detect cst - related peptides .
the middle panel of ( a ) showed the darker exposure of the lower part of the gel .
the right panel of ( a ) showed the running position of the synthetic human cst in the same gel .
for maldi - tof ( b ) , proteins eluted from the beads with acetonitrile / water / tfa were lyophilized and analyzed by mass spec catestatin spanning peptides detected by mass spectrometry from immunoprecipitation of heart extract .
proteins were eluted from the beads with acetonitrile / water / tfa , lyophilized and analyzed by maldi - tof .
to determine the expression of chromogranin / secretogranin proteins , primary culture of cardiomyocytes from 3- to 4-day - old pups were analyzed by immunocytochemistry .
deconvolution microscopy shows punctuate staining of chromogranin / secretogranin proteins , which indicates discrete vesicular distribution of chga ( fig . 
1c ) proteins in the cytoplasm of murine cardiomyocytes . as a positive control , we detected vesicular staining for the myocyte specific protein ( myh : 60% of cells ) ( fig . 
1d ) and atrial natriuretic peptide ( anp : 4% of cells ) ( fig .  1e ) .
cardiomyocyte cell extracts showed several distinct low molecular mass ( ranging from 25 to 37  kda ) cst - positive fragments , along with low abundance full - length chga ( ran at 75  kda ) ( fig . 
the localization of chga ( a ) , chgb ( b ) and scg2 ( c ) were assessed in primary culture of cardiomyocytes , derived from 3- to 4-day - old pups .
cardiomyocytes were stained with primary antibodies specific for each chromogranin / secretogranin proteins and the cardiomyocyte specific markers such as myosin heavy chain ( myh ) ( d ) and atrial natriuretic peptide ( anp ) ( e ) .
the secondary antibodies used were donkey anti - rabbit igg - alex flur 488 ( green fluorescence , 1:250 ) and donkey anti - goat igg - alex flur 594 ( red fluorescence , 1:350 ) .
cells were examined by deconvolution microscopy using a 60 objective ( scale bar 5  m ) for the upper panel and a 20 objective ( scale bar 10  m ) for the lower panel .
f cardiomyocyte cell extracts ( lane 1 20  g protein and lane 2 40  g protein ) were analyzed by sds - page followed by immunoblot with a cst specific antibody .
g freshly frozen adult ( 6-month - old ) mice heart and adrenal tissues were homogenized in tris - maleate buffer and were subjected to sds - page and immunoblot analysis .
heart extracted proteins ( 75  g ) were run by sds - page for the detection of chga ( by rabbit anti - human cst ) , chgb ( by goat anti - human chgb ) and scg2 ( by rabbit anti - human scg2 ) .
adrenal extracts ( proteins in lane 1 2  g , lane 2 15  g , lane 3 30  g , and lane 4 60  g ) were also run by sds - page for the detection of chga ( by rabbit anti - human cst ) .
for adrenal extract the full - length chga signal was shown in the upper panel and the low molecular mass fragments were shown in the lower panel the expression of chromogranin / secretogranin proteins in murine heart .
the localization of chga ( a ) , chgb ( b ) and scg2 ( c ) were assessed in primary culture of cardiomyocytes , derived from 3- to 4-day - old pups .
cardiomyocytes were stained with primary antibodies specific for each chromogranin / secretogranin proteins and the cardiomyocyte specific markers such as myosin heavy chain ( myh ) ( d ) and atrial natriuretic peptide ( anp ) ( e ) .
the secondary antibodies used were donkey anti - rabbit igg - alex flur 488 ( green fluorescence , 1:250 ) and donkey anti - goat igg - alex flur 594 ( red fluorescence , 1:350 ) .
cells were examined by deconvolution microscopy using a 60 objective ( scale bar 5  m ) for the upper panel and a 20 objective ( scale bar 10  m ) for the lower panel .
f cardiomyocyte cell extracts ( lane 1 20  g protein and lane 2 40  g protein ) were analyzed by sds - page followed by immunoblot with a cst specific antibody .
g freshly frozen adult ( 6-month - old ) mice heart and adrenal tissues were homogenized in tris - maleate buffer and were subjected to sds - page and immunoblot analysis .
heart extracted proteins ( 75  g ) were run by sds - page for the detection of chga ( by rabbit anti - human cst ) , chgb ( by goat anti - human chgb ) and scg2 ( by rabbit anti - human scg2 ) .
adrenal extracts ( proteins in lane 1 2  g , lane 2 15  g , lane 3 30  g , and lane 4 60  g ) were also run by sds - page for the detection of chga ( by rabbit anti - human cst ) . for adrenal extract the full - length chga signal
was shown in the upper panel and the low molecular mass fragments were shown in the lower panel to identify cst - containing fragments , we carried out sds - page followed by cst immunoblot in heart and adrenal extracts .
adult heart extracts revealed three cst - positive fragments with masses of 37 , 31 and 15  kda ( fig . 
murine heart also showed faint immunoreactivity at 75  kda , which is the apparent migratory behavior of full - length chga in sds - page gel .
in addition , we detected a very high molecular mass form of chga , possibly representing the proteoglycan form . because of the presence of a high number of acidic amino acid residues within the primary structure of chromogranin / secretogranin proteins , these proteins run anomalously on sds - page and their apparent molecular weight differs from the weight deduced by primary amino acid sequence .
in contrast to chga , chgb showed a major full - length form and also some low molecular mass fragments ( fig . 
scg2 , on the other hand , showed extensive aggregation or modification as evidenced by the presence of high molecular mass protein as major signal along with some low molecular mass fragments .
compared to the heart extract , the full - length chga appeared to be highly prevalent in the adrenal extract ; the major cst positive fragment ran at 75  kda .
after exposing the lower part of the membrane for a longer period of time , we detected comparable processing products of chga between adrenal gland and heart as evidenced by the presence of smaller , processed forms of chga with masses of 37 , 27 , and 15  kda in the adrenal extract ( fig . 
although both the adrenal gland and heart express the processed forms , the ratio of processed to full - length form is much higher in the heart compared to the adrenal gland indicating an extensive processing of chga in the heart as compared to the adrenal gland .
although chga is overexpressed in humans with essential hypertension and in genetic model of rodent hypertension , cst showed diminished expression in hypertensive subjects and in normotensive individuals with a family history positive for hypertension .
since hypertension is a late penetrating disease , and cst plays crucial roles in the development of hypertension , we sought to determine whether processing of chga to cst in murine heart is altered with age .
the full - length chga signal ( ran 75  kda ) as well as the intensity of the 37  kda chga c - terminal fragment were increased in older animals ( fig . 
however , the intensity of a low molecular mass cst - specific peptide ( less than 15  kda ) decreased in the heart of older animals ( fig . 
consistent with immunoblot findings , cst eia displayed a significant reduction of endogenous cst peptide in the heart extract of older mice ( 3090  days old ) as compared to younger ( 10-day - old ) mice ( fig . 
heart from different age groups of mice ( age in days : 10 , 30 , 60 and 90 ) were collected , homogenized and subjected to sds - page and immunoblot using a cst - specific antibody ( rabbit anti - human cst ) ( a ) .
b the endogenous cst concentrations were measured from tissue extracts using a cst eia kit .
cst concentrations are expressed in pg/g of protein age - dependent processing of chga to cst in murine heart .
heart from different age groups of mice ( age in days : 10 , 30 , 60 and 90 ) were collected , homogenized and subjected to sds - page and immunoblot using a cst - specific antibody ( rabbit anti - human cst ) ( a ) .
b the endogenous cst concentrations were measured from tissue extracts using a cst eia kit .
mouse heart extract was purified using the rp - hplc column ( fig .  3 ) and the fractions collected for slot blot analysis . from the anti - cst slot blot , two fractions eluting at 48% ( 6163  min ) and 54% ( 7376  min ) acetonitrile were positive for cst immunoreactivity ( fig . 
3b ) . to evaluate the apparent molecular mass of chga c - terminal cst - containing fragments in these fractions , each fraction was lyophilized , pooled and subjected to mass spec analysis .
electrospray mass spec analysis revealed the presence of several cst - spanning peptides in heart extracts ( table  1 ) . among them , the peptides of mass 5,051.60 and 5,641.11 , representing chga356398 and chga357404 or chga358405 , respectively , had considerable overlap with the n - terminal part of the long cst sequence , mchga356384 .
the peptides of mass 4,757.50 , 5,244.85 and 2,478.34 contained a significant part of the n - terminal of short cst sequence , mchga364384 ( table  1 ) . 

mice hearts ( 6 months old ) were homogenized in acetic acid and subjected to reverse phase hplc purification .
the proteins were eluted ( the chromatogram showing protein elution as measured at 214  nm with time is shown in ( a ) ) , with a gradient of acetonitrile and the fractions were collected , lyophilized and spotted onto nitrocellulose membrane to probe with a cst - specific antibody ( b ) .
fraction numbers are shown on the right of the respective slot blot ( b)table  1catestatin - related peptides detected by mass spectrometry from reverse phase hplc purification of heart extract .
the cst - immuno positive fractions from reverse phase hplc , as detected by slot blot , were lyophilized , and subjected to electrospray mass spectrometry .
the resulting peptides generated in the catestatin region are listed in boldmass ( monoisotopic ) submittedmass ( monoisotopic ) matchedsequencecleavage sites4,989.674,989.37325364e / er / s4,989.54349390k / ep / s5,148.745,148.52328369e / es / f5,057.515,057.52339380k / rp / g3,061.553,061.50342367s / rk / l4,341.314,341.17344380m / dp / g3,034.663,034.56346371q / lr / t4,989.784,989.54349390k / ep / s5,051.65,051.47356398r
/ le / a5,641.115,643.69357404/358405l / e or e / ge / e or e / g3,294.723,294.67360387e / dg / w4,757.54,751.41364403d / rf / e5,244.855,244.63365408r / se / e2,478.342,478.36367387m / kg / w1,754.941,754.87369383l / sq / l2,676.452,676.41369390/370391s / f or l / ss / s or p / s5,187.645,187.51369412l / ss / a2,369.222,369.2376395y / gd / s3,828.983,828.88378409f / re / e2,008.992,009.00379395r / dd / smouse catestatin sequence:[kr]legeddpdrsmklsfrtraygfrdpgpql[rr]long cst : mchga356384short cst : mchga364384 identification of cst - related peptides from heart extract .
mice hearts ( 6 months old ) were homogenized in acetic acid and subjected to reverse phase hplc purification .
the proteins were eluted ( the chromatogram showing protein elution as measured at 214  nm with time is shown in ( a ) ) , with a gradient of acetonitrile and the fractions were collected , lyophilized and spotted onto nitrocellulose membrane to probe with a cst - specific antibody ( b ) .
fraction numbers are shown on the right of the respective slot blot ( b ) catestatin - related peptides detected by mass spectrometry from reverse phase hplc purification of heart extract .
the cst - immuno positive fractions from reverse phase hplc , as detected by slot blot , were lyophilized , and subjected to electrospray mass spectrometry .
the resulting peptides generated in the catestatin region are listed in bold mouse catestatin sequence : [ kr]legeddpdrsmklsfrtraygfrdpgpql[rr ] long cst : mchga356384 short cst : mchga364384
since young animals showed more low molecular mass cst fragments ( < 15  kda ; see fig . 
2a ) , we sought to purify cst related peptides from 1-month - old mice with an alternate strategy of immunoprecipitation .
sds - page analysis ( on a 1020% tricine gel ) of immunoprecipitated complex showed several fragments ranging from 20 to 150  kda ( fig . 
4a , left panel ) . besides these high molecular mass fragments , the longer exposure of the lower part of the membrane revealed the presence of a peptide that ran at 7  kda ( fig .  4a , middle panel ) .
as a positive control , we ran synthetic human cst ( hchga352372 ; molecular mass    2.4  kda ) ( fig . 
although we did not achieve a good separation between 2 and 7  kda protein even on a 1020% gel , our results indicate the presence of a low molecular mass ( in the range of 27  kda ) fragment in the anti - cst immunoprecipitated complex .
the maldi - tof analysis showed a major peptide of 11  kda corresponding to the c - terminal chga derived fragment chga283384 ( fig . 
several peptides with cst motif have been identified . among them , peptides of mass 4,097 and 6,224 overlapped with the n - terminal sequence of short cst , mchga364384 ( table  2 ) . 

mice heart ( 1-month - old animal ) were homogenized in tris - maleate buffer and subjected to immunoprecipitation from 2 to 1  mg protein with anti - cst antibody .
the antigen - antibody complexes were pulled down with protein a / g beads and subjected to immunoblot analysis ( lane 1 ip from 2  mg protein , lane 2 ip from 1  mg protein ) ( a ) . in ( a )
, rabbit anti - human cst antibody was used to detect cst - related peptides .
the middle panel of ( a ) showed the darker exposure of the lower part of the gel .
the right panel of ( a ) showed the running position of the synthetic human cst in the same gel .
for maldi - tof ( b ) , proteins eluted from the beads with acetonitrile / water / tfa were lyophilized and analyzed by mass spectable  2catestatin spanning peptides detected by mass spectrometry from immunoprecipitation of heart extract .
proteins were eluted from the beads with acetonitrile / water / tfa , lyophilized and analyzed by maldi - tof .
the detected peptides are summarized belowmass ( average ) submittedmass ( average ) matchedsequence4,0974,097.65339372/3403734,098.623653994,2284,225.823383725,7335,736.463313775,738.533423895,9615,961.673333816,2246,218.8636541711,92011,920.02283384 anti - cst immunoprecipitation from the heart extract .
mice heart ( 1-month - old animal ) were homogenized in tris - maleate buffer and subjected to immunoprecipitation from 2 to 1  mg protein with anti - cst antibody .
the antigen - antibody complexes were pulled down with protein a / g beads and subjected to immunoblot analysis ( lane 1 ip from 2  mg protein , lane 2 ip from 1  mg protein ) ( a ) . in ( a ) ,
the middle panel of ( a ) showed the darker exposure of the lower part of the gel .
the right panel of ( a ) showed the running position of the synthetic human cst in the same gel .
for maldi - tof ( b ) , proteins eluted from the beads with acetonitrile / water / tfa were lyophilized and analyzed by mass spec catestatin spanning peptides detected by mass spectrometry from immunoprecipitation of heart extract .
proteins were eluted from the beads with acetonitrile / water / tfa , lyophilized and analyzed by maldi - tof .
consistent with previous findings ( pieroni et al . 2007 ; steiner et al . 1990
punctate staining patterns indicate vesicular storage of chromogranin / secretogranin proteins in cardiomyocytes in congruence with the elegant demonstration by winkler s group using immunoelectron microscopy ( steiner et al .
in addition to chga and chgb , we also found scg2 in cardiomyocytes , which has not been shown before .
a recent confocal microscopical study reveals co - localization of chga with bnp in human ventricular myocytes of dilated and hypertrophic hearts ( pieroni et al .
chga has also been detected in purkinje conduction fibers , in both atrium and ventricle , as well as in h9c2 cardiomyocytes ( weiergraber et al .
tota s group was the first to demonstrate the cardiosuppressive effects of the n - terminal chga peptide vst on isolated and perfused heart preparations of eel , frog and rat , and to establish vst s direct involvement in the regulation of the contractile performance of hearts beating under basal ( i.e. , non - stimulated ) as well as stimulated ( e.g. , isoproterenol ) conditions ( cerra et al .
they have detected natural vst - containing peptides in rat heart ( glattard et al .
2006 ) , which basically established an autocrine / paracrine regulation of cardiac function by vst .
we have recently demonstrated that , like vst , cst also induces cardio - suppressive effects in frog and rat heart ( angelone et al .
therefore , it was crucial to demonstrate that cst - containing peptides are in fact generated in the heart . the present studies clearly show that cst - containing peptides are indeed generated in murine heart , which establishes an autocrine / paracrine action of cst in cardiac function .
immunoprecipitation of heart extracts with cst followed by immunoblots confirms generation of cst peptide in heart .
in fact , we have previously shown that cst inhibits catecholamine secretion through an autocrine / paracrine mechanism ( mahata et al .
it is interesting to note that heart extracts or cardiomyocytes contain very little amount full - length chga as opposed to adrenal gland where the major band represents the full - length chga , which is consistent with the findings that catecholamines inhibit proteolytic processing of chga in the adrenal medulla ( wolkersdorfer et al .
the high molecular weight bands detected for chga and scg2 possibly represent the proteoglycan form of these proteins . of note ,
it appears that processing of chga to cst is diminished in heart with advancing age as judged by low cst .
this is particularly relevant in context of hypertension , which is a late - penetrating disease where cst plays a crucial role .
we have previously shown that cst ( low ) is inversely proportional to chga ( high ) in hypertensive human subjects , for which we have implicated a diminished processing of chga to cst ( oconnor et al .
, it should be mentioned that chga has been shown to increase in proportion to severity of cardiac dysfunction and to be associated with prognosis in patients with both chronic and post - infarction heart failure ( ceconi et al .
in addition , it has been shown recently that plasma chga levels obtained within the first 24  h of admission are independently associated with the incidence of death in patients with acute coronary syndromes ( jansson et al .
in conclusion , we have detected chromogranin / secretogranin proteins including chga , chgb and scg2 in murine heart where chga is processed to cst - related peptides , implicating an autocrine / paracrine regulation of cardiac function by cst .
 in the heart , the secretory granules containing the atrial natriuretic peptides ( anp ) and b - type myocardial natriuretic peptide ( bnp ) provide the basis for the endocrine function of this organ . 
 we sought to determine whether atrial and myocardial secretory granules contain chromogranin / secretogranin proteins including chromogranin a ( chga / chga ) , chromogranin b ( chgb / chgb ) and secretogranin ii ( scg2/scg2 ) . 
 deconvolution microscopy on immunolabeled proteins revealed the presence of chga , chgb , and scg2 in murine cardiac secretory granules . 
 the presence of low plasma catestatin ( cst : mchga364384 ) in older mice indicates diminished processing of chga to cst with advancement of age , which is comparable to that found in humans . 
 we have previously shown that cst ( hchga352372 ) exerts potent cardio - suppressive effects on frog and rat heart , but the source of cst for such action has remained elusive . in the present study 
 , we found cst - related peptides in cardiomyocytes and in heart , which establishes an autocrine / paracrine function of cst in cardiac tissue . 
 we conclude that cardiac secretory granules contain chga , chgb and scg2 and that chga is processed to cst in murine heart . 
Introduction
Materials and methods
Results
Expression of chromogranin/secretogranin proteins in murine heart and processing of Chga to CST in heart as compared to adrenal gland
Age-dependent processing of Chga in murine heart
Identification of CST-containing peptides in adult mouse heart
Immunoprecipitation of CST related peptides from young mouse heart
Discussion