Distinct cardiodynamic and molecular characteristics during early and late stages of sepsis-induced myocardial dysfunction

Abstract

We hypothesized that progressive decline in myocardial performance would correlate with upregulation of markers for apoptotic mechanisms following increased duration of polymicrobial sepsis in the rat. Male Sprague-Dawley rats (350-400 g) were randomized into sham, 1-, 3- and 7-day sepsis groups. Each septic rat received 200 mg/kg cecal inoculum intraperitoneally (i.p). The post-mortem analysis showed a severely inflamed peritoneum with the presence of pus in all septic animals that was directly proportional to the duration of sepsis. We observed 10, 33 and 42% mortality in the 1-, 3- and 7-day sepsis groups, respectively. Septic animals at 3 and 7 days exhibited an increased wet lung/total body weight and heart weight/total body weight. A significant increase in total cardiac troponin I (cTnI) and C Reactive Protein (CRP) and endothelin-1 (ET-1) was also observed with an increased duration of sepsis. Myocardial ET-1 concentration in the 7-day post-sepsis group was significantly elevated compared to the sham and 1-day post-sepsis groups. Sepsis also produced a significant decrease in the mean arterial pressure in the 7-day post-sepsis group and tachycardia in the 1-, 3-, and 7-day post-sepsis groups compared to the sham group. A significant prolongation of the left ventricular isovolumic relaxation rate constant, tau, and left ventricular end-diastolic pressure in the 1-, 3- and 7-day post-sepsis groups compared to the sham group was observed. In addition, a significant decrease in the rates of left ventricular relaxation (-dP/dt) and contraction (+dP/dt) in the 3- and 7-day post-sepsis groups compared to the sham and 1-day post-sepsis group was observed. Sepsis produced a significant upregulation in the expression of myocardial TRADD, cytosolic active caspase-3, the Bax/Bcl(2) ratio, and the mitochondrial release of cytochrome C in the 3- and 7-day post-sepsis groups. We observed a progressive increase in the number of TUNEL positive nuclei, cytosolic caspase-3 activation and co-localization of PARP in the nuclei at 1, 3 and 7 days post-sepsis. These data suggest that the progression of sepsis from 1 day to 3-7 days produce distinct cardiodynamic characteristics with a more profound effect during later stages. The sepsis-induced decline in myocardial performance correlates with the induction of myocardial apoptosis.

Figure 1

A Chronic peritonitis as seen during postmortem examination of peritoneal cavity following polymicrobial sepsis in sham, 1-, 3- and 7-days post-septic rat. B. Percentage animals survived at 1, 3 and 7-days post-sepsis. SP-1D, 1-day sepsis; SP-3D, 3-days sepsis; SP-7D, 7-days sepsis.

Figure 2

The effect of sepsis on cardiodynamics in sham, 1-, 3- and 7-day post-sepsis groups. LV isovolumic relaxation rate constant, tau; LV end diastolic pressure, LVEDP; first derivative of LV contraction, +dP/dt and first derivative of LV relaxation, −dP/dt. *p ≤ 0.05 as compared to the sham group, #p ≤ 0.05 as compared to the 1-day sepsis group. SP-1D, 1-day sepsis; SP-3D, 3-days sepsis; SP-7D, 7-days sepsis.

Figure 3

Upper panel exhibits representative blot (from 5 experiments) for the expression of LV procaspase and active caspase-3 in sham, 1-, 3- and 7-days post sepsis groups. Alterations (fold change) in cytosolic active caspase-3/ procaspase ratio normalized to β-actin in sham, 1-, 3- and 7-days post sepsis groups. *p ≤ 0.05 as compared to the sham group, #p ≤ 0.05 as compared to the 1-day sepsis group.

Figure 4

Upper panel exhibits representative blot (from 5 experiments) for the expression LV TNF-α receptor associated death domain (TRADD, Bax and BCl₂ in sham, 1, 3- and 7-days post-sepsis as compared to sham and 1-day post-sepsis. Alterations (fold change) in Bax/ Bcl₂ (lower left panel) and TRADD (lower right panel), normalized to β-actin in sham and septic groups. *p ≤ 0.05 as compared to the sham group, #p ≤ 0.05 as compared to the 1-day sepsis group.

Figure 5

A. Expression of HSP60, β-tubulin in the cytosolic and mitochondrial fractions in LV tissues obtained from sham, 1-, 3- and 7-days post-sepsis as compared to sham and 1-day post-sepsis. B. Representative blot (from 5 experiments) for the expression of Cytochrome C in the cytosolic and mitochondrial fractions of LV tissues. C. Ratio of the expression of cytochrome C normalized to β-actin in the cytosolic and mitochondrial fractions in sham, 1-, 3- and 7-days post-sepsis groups. *p ≤ 0.05 as compared to the sham group.

Figure 6

Representative blot (from 5 experiments) for the expression of LV p38-MAPK, JNK, phosphorylated p38-MAPK (pp38-MAPK) and phosphorylated JNK (pJNK) in sham, 1-, 3- and 7-days post-sepsis groups. Alterations (fold change) in JNK & pJNK (lower panel), and p38-MAPK & pp38-MAPK (middle panel) normalized to β-actin in sham, 1-, 3- and 7-days post-sepsis groups. *p ≤ 0.05 as compared to the sham group, #p ≤ 0.05 as compared to the 1-day sepsis group.

Figure 7

Immunohistochemistry performed in paraffinized LV tissue sections and visualized by using confocal microscopy. A. Representative photomicrographs (magnification 63x) of LV tissue sections from sham, 1-, 3- and 7-day post-sepsis stained for DNA fragmentation using APO-BrdU TUNEL kit (Invitrogen). TUNEL positive cells are stained green (Alexa Flour 488) in blue nuclei (TO-PRO) along with NOMARSKY DIC (Differential Interference Contrast) images of LV tissue sections. B. Representative pictomicrographs (magnification 40x) of LV tissues from sham, 1-, 3- and 7-days post-sepsis stained for caspase-3 expression using FITC-conjugated caspase-3 antibody. The cytosol of the LV tissue is stained green (FITC) and the nuclei are counterstained blue with TO-PRO. C. Representative pictomicrographs (magnifications 200x and 40x) of LV tissues from sham, 1-, 3- and 7-days post-sepsis stained for PARP expression using FITCconjugated caspase-3 antibody. The nucleus of the LV tissue exhibits green (FITC) fluorescence depicting DNA breaks in the blue nuclei which are counterstained with TO-PRO.