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Mechanical unloading improves intracellular Ca²⁺ regulation in rats with doxorubicin-induced cardiomyopathy

Tohru Takaseya, MD^*,*, Masaru Ishimatsu, MD, PhD, Eiki Tayama, MD, PhD^*, Akinori Nishi, MD, PhD, Takashi Akasu, MD, PhD and Shigeaki Aoyagi, MD, PhD^*

^* Department of Surgery, Kurume University School of Medicine, Kurume, Japan
Department of Physiology, Kurume University School of Medicine, Kurume, Japan

View larger version (20K): [in a new window]   Figure 1 Experimental design. Normal rats (6 weeks old) were divided into two groups, control group (intraperitoneal administration of 0.4 ml saline 6 times in 2 weeks) and doxorubicin-treated group (intraperitoneal administration of 2.5 mg/kg body weight 6 times in 2 weeks). Two weeks after the final injection, each group was further divided into two groups according to with or without heterotopic heart transplantation (the heart of each group was transplanted into age-matched normal rat abdomen). Cont0 = control group two weeks after the final intraperitoneal injection; DOX0 = doxorubicin-treated group two weeks after the final intraperitoneal injection; Cont = control group four weeks after the final intraperitoneal injection; DOX = doxorubicin-treated group four weeks after the final intraperitoneal injection; Control+UL = the heart from the control group, which was applied unloading for two weeks; DOX+UL = the heart from the doxorubicin-treated group, which was applied unloading for two weeks. Cont+UL was compared with Cont to evaluate the effects of mechanical unloading on normal hearts; DOX+UL was compared with DOX to evaluate the effects of unloading on the failing myocardium. All four groups (Cont, Cont+UL, DOX, and DOX+UL) were age-matched at the time of in vitro analysis.

View larger version (23K): [in a new window]   Figure 2 Representative traces of cell shortening in isolated cardiomyocytes from four groups, evoked by electrical stimulation under perfusion conditions of 37°C, [Ca2+]0 was 1.8 mmol/l, and the pacing rate was 1.0 Hz. The electrical stimulation was applied at the arrow. (A) Cont. (B) Cont+UL. (C) DOX. (D) DOX+UL. Note that the time courses of cell shortening and relaxation are extremely prolonged in DOX and have recovered in DOX+UL. Abbreviations as in Figure 1.

View larger version (26K): [in a new window]   Figure 3 Representative traces of [Ca2+]i transients in isolated cardiomyocytes from four groups, evoked by electrical stimulation under perfusion conditions of 37°C. [Ca2+]0 was 1.8 mmol/l and pacing rate was 1.0 Hz. The electrical stimulation (stim) was applied at the arrow. (A) Cont. (B) Cont+UL. (C) DOX. (D) DOX+UL. Abbreviations as in Figure 1.

View larger version (27K): [in a new window]   Figure 4 Summary of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2a)/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ryanodine receptor (RyR)/GAPDH mRNA levels. The expression levels of SERCA2a and RyR mRNA were not changed in the Cont and Cont+UL groups. The expression levels of SERCA2a mRNA had recovered in the DOX+UL groups, with no change in RyR levels. Other abbreviations as in Figure 1.

View larger version (26K): [in a new window]   Figure 5 Expression levels of Ca2+-regulatory proteins. (A) Representative immunoblots showing the amounts of SERCA2a, RyR, phospholamban (PLB), and GAPDH protein levels in left ventricular (LV) tissues from four groups. The amounts of SERCA2a (B), RyR (C), and PLB (D) were quantified by the Odyssey Infrared Imaging System, and the data were normalized to values obtained from LV tissues in the control group. Data represent the mean value ± SEM (Cont, n = 6; Cont+UL, n = 5; DOX, n = 6; DOX+UL, n = 5) A.U. = arbitrary unit; other abbreviations as in Figures 1 and 4.

View larger version (143K): [in a new window]   Figure 6 Transmitted electron microscopic images of (A) Cont, (B) Cont+UL, (C) DOX, and (D) DOX+UL. In each image, mitochondria (M), myofibrils (MF), and sarcoplasmic reticulum (SR) (arrowhead) are indicated. Note the loss of myofibrils and swelling of mitochondria and SR observed in doxorubicin-induced cardiomyopathic cells. Few of these changes could be seen in DOX+UL. Other abbreviations as in Figure 1.