Refining the reproducibility of a murine model of stress-induced reversible cardiomyopathy

Abstract

Despite the many advantages of isoproterenol (Iso)-induced models of cardiomyopathy, the extant literature suggests that the reproducibility of the Iso-induced stress cardiomyopathy phenotype varies considerably depending on the dose of Iso used, the mode of administration of Iso (subcutaneous vs. intraperitoneal), and the species of the animal that is being studied. Recently, we have shown that a single injection of Iso into female C57BL/6J mice provokes transient myocardial injury that is characterized by a brisk release of troponin I within 1 h, as well as a self-limited myocardial inflammatory response that is associated with increased myocardial tissue edema, inferoapical regional left ventricular (LV) wall motion abnormalities, and a transient decrease in global LV function, which were completely recovered by day 7 after the Iso injection (i.e., stress-induced reversible cardiomyopathy). Here we expand upon this initial report in this model by demonstrating important sexually dimorphic differences in the response to Iso-induced tissue injury, the ensuing myocardial inflammatory response, and changes in LV structure and function. We also provide information with respect to enhancing the reproducibility in this model by optimizing animal welfare during the procedure. The acute Iso-induced myocardial injury model provides a low-cost, relatively high-throughput small-animal model that mimics human disease (e.g., Takotsubo cardiomyopathy). Given that the model can be performed in different genetic backgrounds, as well as different experimental conditions, the acute Iso injury model should provide the cardiovascular community with a valuable nonsurgical animal model for understanding the myocardial response to tissue injury.NEW & NOTEWORTHY The present study highlights the importance of sexual dimorphism with respect to isoproterenol injury, as well as the importance of animal handling and welfare to obtain reproducible results from investigator to investigator. Based on serial observations of animal recovery (locomotor activity and grooming behavior), troponin I release, and inflammation, we identified that the method used to restrain the mice for the intraperitoneal injection was the single greatest source of variability in this model.

Keywords: inflammation; reproducibility; sex; tissue injury.

Figure 1.

Double-hand method for intraperitoneal injection of isoproterenol. A: mouse is placed on the cage lid using the preferred hand, and the tail is gently grasped and pulled backward by the hand. The mouse is then quickly and gently grasped by the scruff of the neck behind the ears with the thumb and index finger of the other (nonpreferred) hand (see Supplemental Video S1). B: mouse is turned over so that the abdomen is exposed and the head is tilted downward. The tail of the mouse is transferred from the preferred hand to between the palm and the little or ring finger of the nonpreferred hand and then held gently but firmly (see Supplemental Video S1). C: intraperitoneal injection is performed in the lower two abdominal quadrants (just above level of hip) in the midline of the abdomen, to avoid injuring the gut, urinary bladder, or other abdominal organs. D: insulin syringe with a short-tip (12.7 mm) 29-gauge needle is inserted bevel up into the abdomen cavity, with the angle of the syringe 20–30° relative to the horizontal plane of the abdomen.

Figure 2.

Mouse behavioral and hemodynamic responses to single intraperitoneal injection of 300 mg/kg of isoproterenol (Iso). A: percentage of mice with normal, decreased, or no locomotor activity at 10, 30, and 60 min; 2, 6, and 24 h; and days 2 and 3 after intraperitoneal injection of 300 mg/kg of Iso. B: systolic, diastolic, and mean arterial pressures at baseline and at 1, 30, 40, 50, 60, 240, and 300 min after the intraperitoneal injection of 300 mg/kg of Iso in female mice (n = 3 mice/time). C: systolic, diastolic, and mean arterial pressures at baseline and at 1, 30, 40, 50, and 60 min after the intraperitoneal injection of phosphate-buffered saline (PBS) in female mice (n = 3 mice/time). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Characterization of stress-induced cardiomyopathy model in male and female mice. A: Kaplan–Meier survival curves of male and female mice treated with isoproterenol (Iso) or phosphate-buffered saline (PBS). B: serum troponin I levels at baseline, day 2, and day 7 in Iso-treated male (n = 8–14 mice/time) and female (n = 8–10 mice/time) mice. C: flow cytometric analysis of the number of CD45⁺ cells/mg of heart tissue at baseline and at day 2 and day 7 after the Iso injection (male, n = 8–11 mice/time; female, n = 11–17 mice/time). *P < 0.05; **P < 0.01; ***P < 0.001, for comparisons between male and female mice; †P < 0.05 and ††P < 0.01, for changes in male and female mice relative to baseline.

Figure 4.

Sexual dimorphic differences in the myocardial inflammatory response following the isoproterenol (Iso) injection. Flow cytometry was performed on CD45⁺ cells that were isolated from the heart (cells/mg of tissue) at baseline and at day 2 and day 7 after the Iso injection (male, n = 8–11 mice/time; female, n = 10–14 mice/time). A–F: Ly6G⁺ neutrophils (A), Ly6C^highCD64^low monocytes (B), CD64⁺Ly6C^low/− macrophages (C), CD4⁺ T cells (D), CD8⁺ T cells (E), and CD19⁺ B lymphocytes (F). To reduce the number of mice euthanized, a proportion of the male (n = 8 or 9) and female mice (n = 8–13) shown in A–F were reproduced from a supplemental data file in a prior publication (1). *P < 0.05; **P < 0.01; ***P < 0.001, for comparisons between male and female mice; †P < 0.05; ††P < 0.01; †††P < 0.001, for changes in male and female mice relative to baseline.

Figure 5.

Echocardiographic assessment of left ventricular (LV) structure, function, and regional wall motion in male and female mice. A: regional segmental wall motion from LV base to apex in male and female mice at baseline and at 1 h, 6 h, and 7 days after isoproterenol (Iso) injection (male, n = 7 or 8 mice/time; female, n = 5–12 mice/time). B: segmental wall motion score index (SWMSI) in 12 radial segments at baseline and at 1 h, 6 h, and 7 days after the Iso injection (male, n = 7 or 8 mice/time; female, n = 5–12 mice/time). C: global SWMSI at baseline and at 1 h, 6 h, and 7 days after the Iso injection (male, n = 7 or 8 mice/time; female, n = 5–12 mice/time). The global SWMSI was determined as the average of 84 regional LV segments, where 1 = normal wall thickening, 2 = hypokinesis, and 3 = akinesis. D and E: LV end-diastolic volume index (LVEDVI; D) and LV ejection fraction (LVEF; E; male, n = 7 or 8 mice/time; female, n = 5–12 mice/time). Comparative data for female mice shown in A–E were taken from a prior publication to reduce the number of mice euthanized (1). **P < 0.01, for comparisons between male and female mice; †P < 0.05; ††P < 0.01; †††P < 0.001, for changes in male and female mice relative to baseline.