Nitrosative Stress as a Modulator of Inflammatory Change in a Model of Takotsubo Syndrome

Figures

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  Figure 1. Functional Effects of ISO Alone Echocardiographic strain data derived from apical myocardial motion in (A) pre-treated state and (B) 24-h post-injection of isoproterenol (ISO). Post-ISO analysis showed marked impairment of apical radial strain. Ant = anterior; AntSept = anteroseptal; AVC = aortic valve closure; Inf = inferior; Lat = lateral; Post = posterior; SAX-AP = short axis-apex; Sept = septal; SR = strain rate.

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  Figure 2. Effects of ISO on Inflammatory Markers Leukocyte infiltration, as measured by CD45, is shown in (A) control rat myocardium and (B) isoproterenol (ISO)-treated myocardium, with (C) summary data. Macrophage infiltration, measured with CD68 expression, is shown in (D) control rat myocardium and (E) ISO-treated myocardium, with (F) summary data. Five sections from each group were analyzed (20 sections in total); black/dark brown represents positive staining. *p < 0.05.

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  Figure 3. Immunohistochemical Results for ISO Alone (Summative Data) Effects of isoproterenol (ISO) on regional accumulation of 3-nitrotyrosine (3-NT), poly(ADP-ribose) (PAR), and thioredoxin-interacting protein (TXNIP) in left ventricular myocardium. (A) 3-NT mean values at apex and base. Two-way analysis of variance (ANOVA): ISO, p = 0.002; apex/base, p = 0.03; and interaction, p = 0.23. (B) PAR mean values at apex and base. Two-way ANOVA: ISO, p < 0.0001; apex/base, p = 0.01; and interaction, p = 0.93. (C) TXNIP mean values at apex and base. Two-way ANOVA: ISO, p < 0.0001; apex/base, p = 0.002; and interaction, p = 0.57. Two slides were analyzed per rat (apex and base); values are mean ± SEM. *p < 0.05.

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  Figure 4. Immunohistochemical Effects of ISO Alone (Representative Images) Effects of ISO on 3-NT, TXNIP, and PAR expression in the myocardium. The accumulation of 3-NT is shown in (A) control rat myocardium and (B) ISO-treated myocardium. TXNIP accumulation is shown in (C) control rat myocardium and (D) ISO-treated myocardium. PAR accumulation is shown in (E) control rat myocardium and (F) ISO-treated myocardium. Abbreviations as in Figure 3.

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  Figure 5. Immunoblotting Results for 3-NT These demonstrate a significant increase in the 47-kDa band (p = 0.003) and a borderline increase in the 70-kDa band (p = 0.05) (2-way ANOVA) with ISO treatment without statistically significant regional variability between apex and base. Five control rats were compared with 5 ISO-treated rats, with blots completed in triplicate. Western blots; CA = control apex; CB = control base; IA = ISO apex; IB = ISO base; other abbreviations as in Figure 3.

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  Figure 6. Functional Effects of ISO/3AB Changes in left ventricular apical radial strain over 24 h after ISO (solid line) or ISO/3-aminobenzamide (3AB) (dotted line). Two-way ANOVA: interaction, p = 0.0004; treatment, p < 0.0001; and time, p < 0.0001. Values are mean ± SEM. *p < 0.05. Abbreviations as in Figure 3.

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  Figure 7. Immunohistochemical Results for ISO Versus ISO/3AB (Summative Data) Effects of ISO on regional accumulation of 3-NT, PAR, and TXNIP in left ventricular myocardium with 3AB (ISO/3AB) and without (ISO). (A) 3-NT, showing mean values at apex and base. Two-way ANOVA: 3AB, p = 0.44; apex/base, p = 0.17; and interaction, p = 0.18. (B) PAR mean values at apex and base. Two-way ANOVA: 3AB, p = 0.0001; apex/base, p = 0.0043; and interaction, p = 0.86. (C) TXNIP mean values at apex and base. Two-way ANOVA: 3AB, p < 0.0001; apex/base, p = 0.57; and interaction, p = 0.26. Two slides were analyzed per rat (apex and base); values are mean ± SEM. *p < 0.05. Abbreviations as in Figures 3 and 6.

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  Figure 8. Immunohistochemical Effects of ISO Versus ISO/3AB (Representative Images) Effects of ISO on 3-NT, TXNIP, and PAR expression in the myocardium, in the presence of ISO and in the absence of the poly(ADP-ribose) polymerase-1 inhibitor 3AB (ISO/3AB). 3-NT accumulation is shown in (A) ISO-treated myocardium and (B) ISO/3AB-treated myocardium. TXNIP accumulation is shown in (C) ISO-treated myocardium and (D) ISO/3AB-treated myocardium. PAR accumulation is shown in (E) ISO-treated myocardium and (F) ISO/3AB-treated myocardium. Abbreviations as in Figures 3 and 6.

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  Figure 9. Proposed TS Signal Transduction Cascade ISO injection results in β-adrenoceptor activation, leading to activation of coupled endothelial nitric oxide synthase (eNOS) receptors and to mitochondrial reactive oxygen species release. With increased superoxide (O2–) and nitric oxide (NO) production, there is formation of peroxynitrite (ONOO–), leading to protein nitration, myocardial inflammation, and poly(ADP-ribose) polymerase-1 (PARP-1) activation, resulting in energetic impairment as well as PAR production. Inflammatory activation of glycocalyx shedding contributes to the development of edema and to increased expression of TXNIP. ATP = adenosine triphosphate; BNP = B-type natriuretic peptide; DNA = deoxyribonucleic acid; iNOS = inducible nitric oxide synthase; NAD+ = nicotinamide adenine dinucleotide; other abbreviations as in Figure 3.