Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Abstract

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

Figure 1

Doxo induces myofibrillar structural damage in ARVM. Cells were cultured for 12 days and then treated with 1 µM of Doxo for 48 h. ARVM were stained for all actin using rhodamin-phalloidin (red) and immunostained for the M-line protein myomesin (green). Left: untreated ARVM. Right: structural damage was heterogeneously distributed in the cultures treated with Doxo including the presence of virtually unchanged cells (green arrow) and others losing all sarcomeric structure (red arrow). Cells from 4 hearts were independently treated and analyzed.

Figure 2

Effect of chronic SNP treatment on contractility and calcium. Freshly isolated cardiomyocytes were cultured in serum-free medium and treated with the all-NOS inhibitor L-NMMA 100 µM or sodium nitroprusside as indicated for 18 h before measurement at 37°C and with 2 Hz electrical field pacing. SNP at 50 and 100 µM caused a significant reduction of fractional shortening (left) and of the late phase of relaxation, but no significant change of systolic fura-2 ratio (right) or its kinetics in the late phase of relaxation. L-NMMA did not induce significant changes in any of the measured parameters. Typical contraction and fura2-transients are shown below for each treatment. Black arrows below the time axis indicate the pacing events. *P<0.05 vs untreated, **P<0.001 vs untreated, n = 20 cells from 3 hearts.

Figure 3

Viability and myofibrillar structure. Isolated ARVM were cultured for 14 days then treated with SNP or Doxo and cell viability and structural changes were analyzed. A) Left: assay for caspase-3 activity after treatment with 3 mM SNP or 10 µM Doxo for 6 h. Right: MTT assay after treatment with different concentrations of SNP or a pan-caspase inhibitor for 12 hours. B, C) Cultures were treated with 3 mM SNP for 0–15 h and processed with the TUNEL assay (positive nuclei: green), then immunostained for actin (red) and alpha-actinin (blue) and counted. The * and # signs in (B) indicate the time points, when results became significant vs untreated cells at the level of P<0.05 and better in all following measurements. Representative confocal images as shown in (C), 20 fields in each condition, were used for assessing the percentage of cells displaying morphological changes. * and # P<0.05 vs untreated, ***P<0.001 vs untreated, cells were isolated from 3 hearts and independently processed.

Figure 4

SNP gradually affects myofibrillar integrity over time. Isolated ARVM were cultured for 14 days then treated with 3 mM SNP for 3 and 12 h. Typical cells are shown out of 15 photographed areas for each condition from 3 independently processed hearts. ARVM were stained with rhodamin-phalloidin for all actin (A1–C1) and immunostained for α-actinin (A2–C2). Magnified inserts in the α-actinin column demonstrate the gradual degradation of sarcomeric structure including clumping of myofibrils and contraction of the cells.

Figure 5

SNP alters the ultra-structure of cardiomyocytes. Isolated ARVM were cultured for 14 days then treated with 3 mM SNP for 0 (A), 3 (B) and 5 (C) hours and processed for electron microscopy. Typical cells are shown out of 15 areas for each condition from 3 independently processed hearts. Mitochondrial swelling was observed in SNP-treated cultures. Typical mitochondria are indicated with black arrowheads (A–C). Fasciae adherens and desmosomes are visible in the middle of image C (black asterisks) indicating a line of cell-cell contacts between two cardiomyocytes differently affected by the SNP treatment.