At the beginning of the protocol, body weight was similar between the groups (~230 ± 15 g). After 90 days of MI, STZ-diabetic rats had a reduced body weight (D: 213 ± 10 and DI: 211 ± 10 g) compared with normoglycemic rats (C: 508 ± 3 and I: 471 ± 9 g). Glycemia was increased in STZ-diabetic rats (D: 400 ± 27 and DI: 371 ± 37 mg/dL) in comparison with normoglycemics (C: 90 ± 3 and I: 96 ± 3 mg/dL).
Left Ventricular Function: Noninvasive and Invasive Evaluations
The myocardial infarction akinetic area (MI area) measured by echocardiography was similar between infarcted groups at the initial (I: 40 ± 3 and DI: 41 ± 3% of LV wall) and final (I: 47 ± 2 and DI: 40 ± 4% of LV wall) evaluations. There was a correlation between the MI area evaluated by echocardiography and Masson's trichrome stain histological method (r = 0.90, p < 0.0001).
The noninvasive left ventricular function parameters are shown in Table . Initial evaluation, performed 2 days after the myocardial infarction surgery, showed that LV mass, IVRT, EDT, and MPI were similar between the experimental groups. In contrast, increased LV chamber (LVEDD), reduced EF, and VCF were observed in the infarcted groups (I and DI) compared with noninfarcted (C and D) rats at the initial evaluation.
Initial and final echocardiographic measurements in control (C), diabetes (D), myocardial infarction (I), and diabetes + myocardial infarction (DI) groups.
Final echocardiographic evaluation (90 days after myocardial infarction, 105 days after STZ induction) showed that LV mass was not different in D rats in relation to the C; however, the I group had an increase compared with the initial evaluation. Moreover, diastolic dysfunction was present in D, I, and DI compared with C rats and in the diabetic groups (D and DI) compared with that at the initial evaluation. The D group had reduced systolic function evaluated by EF and VCF in relation to the initial evaluation and C group (VCF). Myocardial infarction groups (I and DI) demonstrated systolic dysfunction compared with the C group. However, DI animals showed attenuation of systolic dysfunction, as demonstrated by an increase in EF and VCF compared with I rats at the final evaluation. MPI was increased in the final evaluations of the D, I, and DI groups in comparison with the C group. Parallel with these results, DI had reduced MPI compared with the I group, indicating lower global ventricular dysfunction in these animals.
Invasive LV function data, performed after the final echocardiographic evaluation, demonstrated that the LVSP was reduced in D, I, and DI groups compared with that in the C group (92 ± 3, 113 ± 4, 93 ± 4 vs. 134 ± 5 mmHg, respectively). Furthermore, STZ-diabetic animals (D and DI) had additional impairment in LVSP compared with I animals. As expected, LVEDP was markedly increased in both I (20 ± 2 mmHg) and DI (12 ± 3 mmHg) rats compared with D and C rats (6 ± 1 and 5 ± 0.3 mmHg, respectively). However, the DI group had attenuated LVEDP compared with the I group. Ventricular function was also estimated by +dP/dt (inotropic index) and -dP/dt (lusitropic index). Experimental groups (D, I, and DI) exhibited diastolic dysfunction evaluated by -dP/dt compared with the C group (D: -4315 ± 473, I: -3208 ± 481, DI: -4030 ± 484 vs. C: -7186 ± 169 mmHg/sec). Similarly, D, I, and DI animals displayed a reduction in +dP/dt inotropic index (6567 ± 415, 4301 ± 457 and 5702 ± 325 mmHg/sec, respectively) compared with C animals (9445 ± 420 mmHg/sec). However, it is important to point out that the DI group had attenuated systolic dysfunction, also evidenced by invasive measurements (+dP/dt), compared with I rats.
Invasive LV function data paralleled echocardiography findings, as observed by the positive correlation obtained between LVEDD and LVEDP (r = 0.85; p < 0.0005), suggesting that increased diastolic diameter was associated with higher LVEDP values. In addition, a positive correlation was also obtained between EF and +dP/dt (r = 0.82; p = 0.0007).
Hemodynamic and Autonomic Function
Hemodynamic and autonomic evaluations are presented in Table . Diabetic groups (D and DI) had a reduction in mean arterial pressure (MAP) and heart rate (HR) compared with that in nondiabetic groups (C and I). In addition, D, I, and DI animals had a reduction in systolic arterial pressure in comparison with the C group; however, STZ-diabetic (D and DI) rats evidenced an additional reduction of this parameter compared to C and I. Baroreflex sensitivity, evaluated by tachycardic (TR) and bradycardic responses (BR), was worsened in all experimental groups in comparison with C. Therefore, D and DI displayed an additional reduction in BR compared with C and I rats, as observed in the Figure .
Hemodynamic measurements and pulse interval variability in control (C), diabetes (D), myocardial infarction (I), and diabetes + myocardial infarction (DI) groups.
Figure 1 Baroreflex sensitivity estimated by bradycardic (BR) and tachycardic responses (TR) from control (C), diabetes (D), myocardial infarction (I) and diabetes + myocardial infarction (DI) rats (n = 8 for each group). * p < 0.05 vs. C; † p (more ...)
Pulse interval variability, evaluated in time domain by standard deviation of pulse interval (SD), was reduced in all experimental animals compared with C, with additional impairment in the DI group compared with that in D and I (Table ).
Expression of Regulatory Proteins Involved in Intracellular Calcium Homeostasis
GAPDH protein levels remained unchanged among all groups studied and were used to normalize regulatory protein expression involved in calcium homeostasis. SERCA2 expression levels were reduced in both infarcted groups (I and DI) compared with C and D (Figures and ). In addition, positive correlations were found between SERCA2 and EF (r = 0.85; p < 0.001) and SERCA2 and +dP/dt (r = 0.87; p < 0.001). NCX expression levels were increased in D and I groups compared with C; however, DI rats had NCX expression levels reduced in comparison with that in D and I rats (Figures and ). The SERCA2/NCX ratio was reduced in D and I animals in comparison with C, and increased in DI in comparison with D and I animals (Figure ).
Figure 2 Expression levels of regulatory proteins related to intracellular calcium homeostasis from control (C), diabetes (D), myocardial infarction (I), and diabetes + myocardial infarction (DI) rats (n = 8 for each group). Targeted bands were normalized to cardiac (more ...)
We additionally evaluated the expression of PLN, a phosphoprotein that regulates the apparent calcium affinity of SERCA2, and phosphorylated PLN in both Ser16 and Thr17 residues. PLN expression levels were increased in group I compared with that in D, and reduced in DI compared with that in C, D, and I groups (Figures and ). Additionally, the SERCA2/PLN ratio, an important index of sarcoplasmic reticulum calcium uptake capacity, was reduced in I animals compared with C, D, and DI animals (Figure ). The expression levels of both phospho-ser16-PLN and phospho-thr17-PLN were increased only in DI animals compared with C, D, and I rats (Figures and ), indicating higher activation of SERCA2 pump and better calcium reuptake by sarcoplasmic reticulum in this group. PP1 expression, a PLN phosphorylation regulator, was increased in the D group compared with that in C; however, although the increase observed in I and DI was discrete, no statistical changes were evidenced (Figures and ).
Figure 3 Expression levels of intracellular calcium efflux mediators from control (C), diabetes (D), myocardial infarction (I), and diabetes + myocardial infarction (DI) rats (n = 8 for each group). Targeted bands were normalized to cardiac GAPDH. A) Representative (more ...)
Total mortality rate evaluation (Kaplan-Meier survival curve) showed that groups I (13 deaths among 21 animals, 62% of mortality) and DI (22 deaths among 30 animals, 73% of mortality) had a higher mortality rate compared with C (no deaths) and D groups (8 deaths among 16 animals, 50% of mortality). However, the DI group had an increased mortality rate compared with I group.