diabetes mellitus is an important public health issue due to its high prevalence and increased morbidity and mortality .
cardiac injury is caused by coronary atherosclerosis and diabetes - related cardiomyopathy . as first reported by rubler et al .
, diabetic cardiomyopathy is a single form of heart disease characterized by left ventricular systolic and diastolic dysfunction in the absence of underlying coronary artery disease and/or hypertension .
diabetic cardiomyopathy is a common cardiac condition affecting both type 1 and type 2 diabetes patients .
the pathophysiology of diabetic cardiomyopathy is not completely understood as several mechanisms can be involved ; these include myocyte hypertrophy , myocardial fibrosis , contractile dysfunction , calcium handling and mitochondrial function changes , and nitric oxide signaling impairment [ 38 ] .
hyperglycemia - induced oxidative stress is an important factor involved in diabetic cardiomyopathy [ 912 ] .
regular physical exercise is an established nonpharmacological strategy used as an adjuvant therapy in heart failure from different etiologies [ 13 , 14 ] .
clinical studies in stable chronic heart failure have shown that long - term moderate physical training attenuates abnormal cardiac remodeling and improves functional capacity , exercise duration , and quality of life [ 1518 ] . in different cardiac injury models , exercise has been shown to attenuate left ventricular dilatation , myocyte hypertrophy , myocardial fibrosis , mitochondrial dysfunction , myocyte calcium handling changes , sympathoexcitation alterations , cardiac dysfunction , and inflammatory activation [ 1925 ] . in diabetes ,
physical exercise reduces cardiovascular risk factors and improves glycemic control , functional capacity , and muscle strength [ 2629 ] .
however , most clinical studies in diabetes have been performed on type 2 diabetes patients [ 28 , 30 ] . in experimental studies on rats with streptozotocin - induced diabetes
, regular physical exercise has been shown to improve myocardial glucose homeostasis , endogenous antioxidant defenses , cardiac function , heart tolerance to ischemia , and ultrastructural extracellular matrix and mitochondrial changes [ 3133 ] .
in vivo evaluation of cardiac function is subjected to myocardial function modulation by hemodynamic and systemic metabolic abnormalities . left ventricular isolated papillary muscle preparations allow us to properly control preload and afterload and analyze intrinsic myocardial function without the effects of systemic metabolic changes [ 3436 ] .
furthermore , by using positive inotropic stimulation , it is also possible to evaluate myocardial contractile reserve in papillary muscle preparations [ 37 , 38 ] .
therefore , in this study we evaluated the influence of a low intensity aerobic exercise protocol on in vivo cardiac remodeling and in vitro myocardial function in rats with streptozotocin - induced diabetes mellitus .
as oxidative stress is associated with diabetes cardiomyopathy and can be influenced by physical exercise , we also analyzed myocardial oxidative stress in diabetic rats .
male wistar rats were purchased from the central animal house at botucatu medical school , unesp .
all animals were housed in a room under temperature control at 23c and kept on a 12-hour light / dark cycle .
all experiments and procedures were approved by botucatu medical school ethics committee , unesp , botucatu , sp , brazil . the rats were assigned into four groups : sedentary control ( c - sed , n = 14 ) ; exercised control ( c - ex , n = 15 ) ; sedentary diabetes ( dm - sed , n = 25 ) ; and exercised diabetes ( dm - ex , n = 25 ) .
louis , mo , usa ) at the dose of 50  mg / kg diluted in 0.01  m citrate buffer ph 4.5 [ 9 , 3941 ] .
seven days after streptozotocin administration , blood glucose was measured by glucometer ( advantage ) ; only rats with glycemia > 220  mg / dl were considered diabetic and included in the study . after diabetes confirmation , exercise protocol was started in a treadmill ( avs projects , so paulo , brazil ) as previously described .
the protocol was applied once a day , five days a week , for nine weeks .
after the first week of animals adaptation ( 8  min , 8  m / min ) , exercise duration and speed were increased gradually up to 18  min / day at 11  m / min . in the first two weeks of training , the animals were subjected to low - voltage electrical stimulation to start the exercise .
systolic blood pressure was measured at the end of experiment by the tail - cuff method   using an electrosphygmomanometer ( narco bio - system , model 709 - 0610 , international biomedical inc .
echocardiographic evaluation was performed using a commercially available echocardiograph ( general electric medical systems , vivid s6 , tirat carmel , israel ) equipped with a 511.5  mhz multifrequency probe .
rats were anesthetized by intraperitoneal injection of a mixture of ketamine ( 50  mg / kg ) and xylazine ( 0.5  mg / kg )
. a two - dimensional parasternal short - axis view of the left ventricle ( lv ) was obtained at the level of the papillary muscles .
m - mode tracings were obtained from short - axis views of the lv at or just below the tip of the mitral - valve leaflets and at the level of the aortic valve and left atrium [ 4346 ] .
m - mode lv images were printed on a black - and - white printer at a sweep speed of 200  mm / s .
all lv structures were manually measured by the same observer ( ko ) according to the leading - edge method of the american society of echocardiography .
the measurements obtained were the mean of at least five cardiac cycles on the m - mode tracings .
the following structural variables were measured : left atrium ( la ) diameter , lv diastolic and systolic diameters ( lvdd and lvsd , resp . ) , lv diastolic posterior wall thickness ( pwt ) , and aortic diameter ( ao ) .
lv relative wall thickness ( rwt ) was calculated by the following formula : 2  pwt / lvdd . left ventricular mass ( lvm ) was calculated using the following formula : [ ( lvdd + pwt + swt)   ( lvdd) ]  1.04 .
left ventricular function was assessed by the following parameters : endocardial fractional shortening ( efs ) , ejection fraction ( ef ) , posterior wall shortening velocity ( pwsv ) , early - to - late diastolic mitral inflow velocities ratio ( e / a ratio ) , and e wave deceleration time ( edt ) .
a joint assessment of diastolic and systolic lv function was performed by the myocardial performance index ( tei index ) .
two days after the echocardiographic study , intrinsic myocardial contractile performance was evaluated in isolated lv papillary muscle preparation as previously described [ 4750 ] .
rats were anesthetized ( sodium pentobarbital intraperitoneally , 50  mg / kg ) and decapitated .
lv anterior or posterior papillary muscle was dissected free , mounted between two spring clips , and placed vertically in a chamber containing krebs - henseleit solution at 28c and oxygenated with a mixture of 95% o2 and 5% co2 ( ph 7.38 ) .
the composition of the krebs - henseleit solution in mm was as follows : 118.5 nacl , 4.69 kcl , 1.25 cacl2 , 1.16 mgso4 , 1.18 kh2po4 , 5.50 glucose , and 25.88 nahco3 .
the spring clips were attached to a kyowa model 120t-20b force transducer and a lever system which allowed for muscle length adjustment .
after a 60-minute period , during which the preparations were permitted to shorten whilst carrying light loads , muscles were loaded to contract isometrically and stretched to the apices of their length - tension curves ( lmax ) . after a 5-minute period , during which preparations performed isotonic contractions ,
muscles were again placed under isometric conditions and length - tension curve apex was determined .
the following parameters were measured from isometric contraction : peak of developed tension ( dt , g / mm ) , resting tension ( rt , g / mm ) , time to peak of tension ( tpt , ms ) , maximum rate of tension development ( + dt / dt , g / mm / s ) , and maximum rate of tension decline ( dt / dt , g / mm / s ) .
to evaluate contractile reserve , mechanical papillary muscle performance was evaluated at basal condition and after the following inotropic stimulation : postrest contraction , extracellular ca concentration increase , and beta - adrenergic agonist isoproterenol addition to the nutrient solution .
papillary muscle cross - sectional area ( csa ) was calculated from muscle weight and length by assuming cylindrical uniformity and a specific gravity of 1.0 .
papillary muscles with csa > 1.7  mm were excluded from analysis as muscles with csa > 1.7  mm can present central core hypoxia and impaired functional performance . after dissecting papillary muscle , atria and ventricles were separated and weighed .
fragments of lung and left and right ventricles were weighed before and after drying sessions ( 65c for 72  h ) to evaluate the wet - to - dry weight ratio .
five - micrometer - thick sections were stained with hematoxylin and eosin . from each lv ,
the smallest transverse diameter was measured in at least 50 myocytes , in which the nucleus could be clearly identified .
measurements were performed using a leica microscope ( magnification 40x ) attached to a video camera and connected to a computer equipped with image analysis software ( image - pro plus 3.0 , media cybernetics , silver spring , md , usa ) . to estimate myocardial collagen content ,
hydroxyproline concentration was measured in lv tissue as previously described [ 51 , 52 ] .
briefly , the tissue was dried using a speedvac concentrator sc 100 attached to a refrigerated condensation trap ( trl 100 ) and vacuum pump ( vp 100 , savant instruments , inc . , farmingdale , ny , usa ) .
tissue dry weight was measured and the samples were hydrolyzed overnight at 100c with 6  n hcl ( 1  ml/10  mg dry tissue ) . a 50  l aliquot of the hydrolysate was dried in the speedvac concentrator ; 1  l of deionized water was added ; and the sample transferred to a teflon tube .
one milliliter of potassium borate buffer ( ph 8.7 ) was added to maintain constant ph and the sample was oxidized with 0.3  ml of chloramine t solution at room temperature for 20  min .
the addition of 1  ml of 3.6  m sodium thiosulfate and thorough mixing for 10  s stopped the oxidative process .
the tubes were capped and heated in boiling water for 20  min . after cooling to room temperature , the aqueous layer was extracted with 2.5  ml of toluene ; 1.5  ml of toluene extract was transferred to a tube ; and 0.6  ml of ehrlich 's reagent was added .
deionized water and 20  g / ml hop were used as the blank and standard , respectively .
biochemical parameters were measured in serum using a spectrophotometer from pharmacia biotech ( ultrospec 2000 , cambridge , england ) .
analyses were performed with a celm kit ( modern laboratory equipment company , so paulo , brazil ) .
high - density lipoprotein ( hdl ) concentration was measured after precipitation of vldl and ldl by the sodium phosphotungstate / mg method using an enzymatic colorimetric method that incorporates polyethylene glycol - modified cholesterol ester oxidase .
myocardial lipid hydroperoxide concentration was measured in medium containing methanol 90% ( v / v ) , 250  m ammonium ferrous sulfate , 100  m xylenol orange , 25  mm sulfuric acid , and 4  mm butylated hydroxytoluene .
the solution was incubated for 30  min at room temperature and measured at 560  nm .
glutathione peroxidase was assayed in 15  l using 0.15  m phosphate buffer , ph 7.0 , containing 5  mm edta , 0.1  ml of 0.0084  m nadph , 4  g of glutathione - reductase , 1.125  m sodium azide , and 0.15  m glutathione reduced form ( gsh ) in a total volume of 0.3  ml .
superoxide dismutase ( sod ) activity was determined using its ability to inhibit reduction of nitroblue tetrazolium , in medium containing 50  mm phosphate buffer ph 7.4 , 0.1  mm edta , 50  m nbt , 78  m nadh , and 3.3  m phenazine methosulfate .
one unit of sod was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition .
enzyme activities were analyzed at 25c using a microplate reader system ( quant - mqx 200 with kc junior software , bio - tek instruments , winooski , vt , usa ) .
catalase activity was determined in a mixture containing 10  mm hydrogen peroxide and sodium phosphate buffer 50  mm , ph 7.0 , in a final volume of 0.3  ml .
catalase unit was defined as the amount of enzyme related to mol h2o2 decomposition in a first order rate constant during 15  s , at 240  nm . all reagents were purchased from sigma ( st . louis , mo , usa ) .
values were expressed as mean  standard deviation or median and 25th and 75th percentiles , according to normal or nonnormal distribution , respectively .
parameters with normal distribution were compared by one way anova followed by the bonferroni post hoc test and nonnormal parameters were compared by kruskal - wallis and the dunn post hoc test ( comparisons of interest : c - ex versus c - sed , dm - sed versus c - sed , and dm - ex versus dm - sed ) .
the final number of animals in each experimental group was 14 in c - sed , 13 in c - ex , 19 in dm - sed , and 18 in dm - ex .
fifteen rats were excluded from the study due to nonadaptation to treadmill exercise or failure to increase glycemia after streptozotocin administration .
initial body weight ( c - sed : 327  23 ; c - ex : 331  13 ; dm - sed : 336  20 ; dm - ex : 340  20  g ; p > 0.05 ) , glycemia ( c - sed : 109  11 ; c - ex : 108  14 ; dm - sed : 103  12 ; dm - ex : 102  12  mg / dl ; p > 0.05 ) , and final systolic blood pressure ( c - sed : 131  18 ; c - ex : 126  13 ; dm - sed : 135  18 ; dm - ex 140  22  mmhg ; p > 0.05 ) did not differ between groups .
final glycemia was higher in diabetic groups than controls and slightly lower in dm - ex than dm - sed ( figure 1 ) . 

final body weight was lower in diabetic groups than controls and in exercised versus sedentary groups .
however , lv - to - body weight ratio was higher in dm than control groups .
atria weight was lower in dm - ex than c - ex and dm - sed . 

c - ex presented lower lv diastolic diameter and higher relative wall thickness than c - sed .
diabetic groups had higher lv diastolic diameter - to - body ratio , left atrial diameter - to - aorta diameter , and lv mass index and lower posterior wall shortening velocity than control groups .
left atrium diameter , in absolute or normalized to aorta diameter values , was lower in dm - ex than in dm - sed . 

table 3 shows lv papillary muscle functional data . in basal condition , + dt / dt was lower in c - ex than c - sed .
after postrest contraction , + dt / dt was lower in c - ex and dm - sed than c - sed ; dt and dt / dt were lower in dm - sed than c - sed .
after extracellular calcium concentration increase to 2.5  mm , tpt was greater in dm - sed than c - sed .
after isoproterenol addition , dm - sed presented increased tpt and reduced + dt / dt and dt / dt compared to c - sed ; dm - ex had lower dt / dt than c - ex .
myocardial hydroxyproline concentration ( c - sed : 2.06  0.50 ; c - ex : 1.96  0.09 ; dm - sed : 2.19  0.40 ; dm - ex : 2.29  0.36  mg / g ; p > 0.05 ) and lv myocyte diameter ( c - sed : 12.7  0.90 ; c - ex : 13.8  1.62 ; dm - sed : 13.1  1.08 ; dm - ex : 13.7  0.69  m ; p > 0.05 ) did not differ between groups .
total cholesterol concentration was higher in both dm groups than controls and lower in dm - ex than dm - sed ( c - sed : 90.5 ( 86.5100 ) ; c - ex : 98 ( 86.5121 ) ; dm - sed : 188 ( 182198 ) ; dm - ex : 148 ( 136165 )  mg / dl ; p < 0.05 ) .
hdl - cholesterol was lower in dm - sed than c - sed and dm - ex ( c - sed : 41.6 ( 37.849.0 ) ; c - ex : 38.6 ( 37.145.3 ) ; dm - sed : 21.5 ( 20.824.5 ) ; dm - ex : 40.8 ( 30.443.8 ) mg / dl ; p < 0.05 ) .
lipid hydroperoxide concentration was higher in c - ex and dm - sed than c - sed and lower in dm - ex than dm - sed .
catalase and superoxide dismutase activity was lower in dm groups than their respective controls and higher in dm - ex than dm - sed .
glutathione peroxidase activity was lower in c - ex and dm - sed than c - sed and higher in dm - ex than dm - sed .
in this study , we evaluated the effects of a low intensity physical exercise protocol on cardiac remodeling and myocardial oxidative stress and function in type 1 diabetic rats .
we used a low intensity exercise protocol for nine weeks including one week of adaptation .
low intensity exercise protocols have been used in diabetic rats as they may not tolerate more extensive or intense periods of exercise [ 42 , 54 , 55 ] .
low intensity appeared more effective than high intensity exercise training to reduce diabetes - related inflammation in serum and skeletal muscles . in our study ,
the low intensity exercise protocol was sufficient to reduce myocardial oxidative stress and to improve cardiac remodeling and myocardial function . as expected , diabetic animals presented heightened glycemia increase and reduced body weight and physical exercise slightly attenuated serum glucose increase in dm - ex group .
as blood pressure did not differ between groups , we can discard the influence of hemodynamic changes on exercise - induced cardiac changes .
transthoracic echocardiogram was performed to analyze in vivo cardiac structures and left ventricular function . in the c - ex group ,
physical exercise induced a slight reduction in absolute lv diastolic diameter values and an increase in lv relative wall thickness with unchanged systolic or diastolic function .
dm - sed presented lv hypertrophy with dilated left cardiac chambers , characterized by increased lv mass index , lv diastolic diameter - to - body weight ratio , and left atrial diameter , with reduced systolic function , characterized by decreased lv posterior wall shortening velocity , compared to c - sed .
a similar echocardiographic pattern of dilated cardiomyopathy has often been observed in experimental diabetes mellitus [ 5761 ] .
the fact that posterior wall shortening velocity was the only changed functional variable suggests that cardiac function was evaluated early during diabetes cardiomyopathy development .
the higher lung wet - to - dry weight ratio in dm - sed suggests the presence of pulmonary congestion and reinforces the lv dysfunction in dm - sed .
myocardial function analysis in lv papillary muscle preparations showed lower developed tension in postrest contraction ; lower + dt / dt in basal condition , postrest contraction , and after isoproterenol stimulation ; lower dt / dt in postrest contraction and after isoproterenol stimulation ; and higher time to peak tension after calcium and isoproterenol stimulation in dm - sed compared to c - sed .
these results allow us to conclude that diabetes - induced systolic and diastolic myocardial dysfunction is responsible for the echocardiographic pattern of dilated cardiomyopathy observed in the in vivo evaluation .
the echocardiographic study revealed that left atrium diameter was lower in dm - ex than dm - sed .
left atrium dilation is often caused by an increase in lv diastolic pressure , which may result from alterations in lv diastolic and/or systolic function . in clinical studies ,
in fact , an increase in left atrium diameter can be considered an early indicator of diastolic dysfunction .
physical exercise attenuated myocardial dysfunction as dm - ex had papillary muscle parameter values between those of dm - sed and c - ex but not significantly different from either group , except for lower dt / dt after isoproterenol stimulation than c - ex .
in dm - ex , the pulmonary congestion marker ( wet / dry lung weight ratio ) also presented values between those of dm - sed and c - ex . we can therefore conclude that exercise attenuated lung congestion , left atrium dilation , and systolic and diastolic myocardial dysfunction in diabetic rats .
physical exercise has been previously shown to improve cardiac function in diabetic rats and attenuate diabetes - induced impaired fractional shortening , contraction velocity , and relaxation time in isolated cardiomyocytes [ 55 , 63 ] . in diabetes
, several myocardial changes can be involved in myocardial dysfunction . in this study , we evaluated myocyte hypertrophy , myocardial fibrosis , and oxidative stress as mechanisms potentially involved in the benefits of physical exercise .
myocardial fibrosis was assessed by measuring myocardial concentration of hydroxyproline , an amino acid , and the main component of the collagen molecule which can only be found in small concentrations in a limited number of other proteins .
myocyte hypertrophy , assessed by measuring the lower transverse diameter , also did not differ between groups .
we can therefore conclude that myocardial fibrosis and myocyte hypertrophy are not involved in the cardiac effects of physical exercise .
some authors have previously observed increased myocardial interstitial collagen in streptozotocin - induced diabetes rats [ 33 , 64 , 65 ] and attenuation by physical exercise [ 33 , 65 ] .
different data can result from the different methods used to evaluate myocardial fibrosis such as ultrastructural analysis , protein expression measurement by western blot technique , or morphometric analysis using sirius - red staining .
diabetes increased oxidative stress as lipid hydroperoxide concentration was higher and the activity of antioxidant enzymes superoxide dismutase , catalase , and glutathione peroxidase was lower in dm - sed than c - sed . increased myocardial oxidative stress has previously been reported in diabetic rats [ 59 , 66 ] .
we have previously shown that myocardial oxidative stress in aged diabetic spontaneously hypertensive rats was associated with impaired myocardial and ventricular function .
oxidative stress is a state of imbalance between reactive oxygen species production and endogenous antioxidant capacity .
our data showed that diabetes induced both increased reactive oxygen species production and decreased antioxidant capacity due to reduced antioxidant enzymes activity .
current knowledge indicates that oxidative stress plays a role as a major determinant of the onset and progression of diabetes - associated cardiovascular alterations [ 3 , 8 , 10 ] .
although it has been established that oxidative stress plays a major role in the development of cardiovascular diseases , clinical studies have failed to show improved outcomes after antioxidant supplementation in preventing or treating cardiovascular diseases [ 67 , 68 ] . while the mechanisms responsible for these poor results are not clear , researchers have directed their attention towards nonpharmacological therapy to reduce oxidative stress , with physical exercise attracting great interest .
recent experimental studies have shown that chronic moderate exercise can decrease oxidative stress in skeletal muscles [ 69 , 70 ] . in this study
, we observed that physical exercise prevented an increase in myocardial lipid peroxidation and attenuated a decrease in antioxidant enzymes activity .
additional studies are needed to elucidate the mechanisms involved in exercise - induced decrease in myocardial oxidative stress during diabetes mellitus .
studies have shown that increased oxidative stress jeopardizes myocardial function through mechanisms such as microvascular damage , abnormalities in calcium homeostasis , and endothelial dysfunction .
although a causative role could not be demonstrated in this study , myocardial oxidative stress may have been involved in the exercise - induced improvement in myocardial function and cardiac remodeling in diabetes rats .
our data therefore support the idea of the beneficial role of low intensity exercise in myocardial function and cardiac remodeling in rats with unmanaged type 1 diabetes mellitus . in conclusion ,
low intensity physical exercise attenuates myocardial oxidative stress , lung congestion , left atrium dilation , and myocardial dysfunction in type 1 diabetic rats .
 we evaluated the effects of a low intensity aerobic exercise protocol on cardiac remodeling and myocardial function in diabetic rats . 
 wistar rats were assigned into four groups : sedentary control ( c - sed ) , exercised control ( c - ex ) , sedentary diabetes ( dm - sed ) , and exercised diabetes ( dm - ex ) . 
 diabetes was induced by intraperitoneal injection of streptozotocin . 
 rats exercised for 9 weeks in treadmill at 11  m / min , 18  min / day . 
 myocardial function was evaluated in left ventricular ( lv ) papillary muscles and oxidative stress in lv tissue . 
 statistical analysis was given by anova or kruskal - wallis . 
 echocardiogram showed diabetic groups with higher lv diastolic diameter - to - body weight ratio and lower posterior wall shortening velocity than controls . 
 left atrium diameter was lower in dm - ex than dm - sed ( c - sed : 5.73  0.49 ; c - ex : 5.67  0.53 ; dm - sed : 6.41  0.54 ; dm - ex : 5.81  0.50  mm ; p < 0.05 dm - sed vs c - sed and dm - ex ) . 
 papillary muscle function was depressed in dm - sed compared to c - sed . 
 exercise attenuated this change in dm - ex . 
 lipid hydroperoxide concentration was higher in dm - sed than c - sed and dm - ex . 
 catalase and superoxide dismutase activities were lower in diabetics than controls and higher in dm - ex than dm - sed . 
 glutathione peroxidase activity was lower in dm - sed than c - sed and dm - ex . 
 conclusion . 
 low intensity exercise attenuates left atrium dilation and myocardial oxidative stress and dysfunction in type 1 diabetic rats . 
1. Introduction
2. Methods
3. Results
4. Discussion