Autophagy is Involved in Cardiac Remodeling in Response to Environmental Temperature Change

Abstract

Objectives: To study the reversibility of cold-induced cardiac hypertrophy and the role of autophagy in this process. Background: Chronic exposure to cold is known to cause cardiac hypertrophy independent of blood pressure elevation. The reversibility of this process and the molecular mechanisms involved are unknown. Methods: Studies were performed in two-month-old mice exposed to cold (4°C) for 24 h or 10 days. After exposure, the animals were returned to room temperature (21°C) for 24 h or 1 week. Results: We found that chronic cold exposure significantly increased the heart weight/tibia length (HW/TL) ratio, the mean area of cardiomyocytes, and the expression of hypertrophy markers, but significantly decreased the expression of genes involved in fatty acid oxidation. Echocardiographic measurements confirmed hypertrophy development after chronic cold exposure. One week of deacclimation for cold-exposed mice fully reverted the morphological, functional, and gene expression indicators of cardiac hypertrophy. Experiments involving injection of leupeptin at 1 h before sacrifice (to block autophagic flux) indicated that cardiac autophagy was repressed under cold exposure and re-activated during the first 24 h after mice were returned to room temperature. Pharmacological blockage of autophagy for 1 week using chloroquine in mice subjected to deacclimation from cold significantly inhibited the reversion of cardiac hypertrophy. Conclusion: Our data indicate that mice exposed to cold develop a marked cardiac hypertrophy that is reversed after 1 week of deacclimation. We propose that autophagy is a major mechanism underlying the heart remodeling seen in response to cold exposure and its posterior reversion after deacclimation.

Keywords: autophagy; heart; hypertrophy; metabolism; temperature.

FIGURE 1

Chronic cold induces cardiac hypertrophy and deacclimation reverses this process. (A) Mice were subjected to acute cold (AC; 4°C, 24 h), chronic cold (CC; 4°C, 10 days) or CC followed by acute deacclimation (AD; 21°C, 24 h) or chronic deacclimation (CD; 21°C, 7 days). (B) Heart weight (mg) to tibia length (mm) (HW/TL) ratio in control (CT), AC, CC, AD and CD mice. (C) Quantification of cardiomyocyte area in the left ventricular wall (left) and representative histological sections of H&E-stained hearts, which were used for the determination of cardiomyocyte area (right). Magnification, 20×. Scale bar: 50 μm. (D) mRNA expression levels of the hypertrophy markers, Nppa and Acta1 normalized to Ppia. Data were analyzed by one-way ANOVA. Results are presented as means ± SEM (n = 5 mice/group; *p < 0.05 compared with control mice, and ^# p < 0.05 compared with CC mice). One, two, and three symbols denote p < 0.05, p < 0.01, and p < 0.001, respectively.

FIGURE 2

Cardiac metabolism and fibrosis are altered under cold and deacclimation. Mice were subjected to acute cold (AC; 4°C, 24 h), chronic cold (CC; 4°C, 10 days) or CC followed by acute deacclimation (AD; 21°C, 24 h) or chronic deacclimation (CD; 21°C, 7 days). (A) mRNA expression levels of the fatty acid oxidation-related genes, Pdk4, Cpt1b, and Acadm, and the glucose transporter gene, Glut1 normalized to Ppia. (B) Representative images showing Masson’s trichrome staining (left) and quantification of left ventricular fibrotic areas, calculated as the positively stained areas divided by the total area of the heart section (right). Scale bar: 100 μM. (C) mRNA expression levels of the fibrotic markers, Col3a1 and Tgfb1 normalized to Ppia. Data were analyzed by one-way ANOVA. Results are presented as means ± SEM (n = 5 mice/group; *p < 0.05 compared with control mice, and ^# p < 0.05 compared with CC mice). One, two, and three symbols denote p < 0.05, p < 0.01, and p < 0.001, respectively.

FIGURE 3

Cardiac autophagy is modulated by cold and deacclimation. Mice were subjected to acute cold (AC; 4°C, 24 h), chronic cold (CC; 4°C, 10 days) or CC followed by acute deacclimation (AD; 21°C, 24 h) or chronic deacclimation (CD; 21°C, 7 days). (A) Representative image (left) and quantification (right) of the immuno-blot analysis of LC3b and p62 protein levels in the heart. GAPDH was used as loading control (n = 5 mice/group). (B) Western blot analysis of LC3b protein levels in mice subjected to CC or AD with or without the application of leupeptin (Leup) 1 h before sacrifice. GAPDH was used as loading control (western blot was performed using samples from three different mice per group). (C) Representative transmission electron microscopy images of hearts from mice subjected to CC or AD and treated with or without leupeptin. Orange arrowheads indicate autophagosomes. Scale bar: 2 μm. (n = 5 mice/group). Results are expressed as means ± SEMs. Data were analyzed by one-way ANOVA (A) and two-way ANOVA (B) (*p < 0.05 compared with control [CT] mice; ^& p < 0.05 compared with AD mice). One, two, and three symbols denote p < 0.05, p < 0.01, and p < 0.001, respectively.

FIGURE 4

Cardiac autophagy is blocked by 1 week of hydroxychloroquine (Chlor) treatment during deacclimation. Mice were exposed to cold (CC; 4°C, 10 days) or cold followed by deacclimation (CD; 21°C, 7 days). During deacclimation, mice of the CD group were subjected to daily intraperitoneal (i.p.) injection of vehicle (PBS) or hydroxychloroquine (Chlor) for 1 week. (A) Representative image (left) and quantification (right) of the immunoblot analysis of LC3b, p62, Atg7 and Parkin protein levels in the heart. Ponceau staining (PS) and GAPDH were used as loading control (western-blot was performed using heart samples from five mice in CC, three mice in AD and four mice in AD + Chlor). (B) Expression levels of the autophagy-related genes, Atg7, Ulk1, Lc3b, and Parkin, normalized to Ppia in the myocardium (n = 5 mice/group). Results are expressed as means ± SEMs. Data were analyzed by one-way ANOVA (^# p < 0.05 compared with PBS-treated deacclimated mice). One, two, and three symbols denote p < 0.05, p < 0.01 and p < 0.001, respectively.

FIGURE 5

Reversion of hypertrophy is impaired by blockade of autophagy during deacclimation. Mice were exposed to cold (CC; 4°C, 10 days) or to cold followed by deacclimation (CD; 21°C, 7 days). During deacclimation (CD) animals were subjected to a daily intraperitoneal (i.p.) injection with vehicle (PBS) or hydroxychloroquine (Chlor) for 1-week. (A) Heart weight (mg) to tibia length (mm) ratio in CC and CD mice treated or not with Chlor. (B) Quantification of cardiomyocyte area in the left ventricular wall (left) and representative histological sections of hearts stained with H&E, which were used to determine cardiomyocyte area (right). Magnification, 20×. Scale bar: 50 μm. (C) mRNA expression levels of the hypertrophy marker Nppa and the fibrotic markers Col3a1 and Tgfb1 normalized to Ppia. Data were analyzed by one-way ANOVA. Results are presented as means ± SEM (n = 5 mice/group; *p < 0.05 compared with CC mice, and ^# p < 0.05 compared with PBS-treated CD mice). One, two, and three symbols denote p < 0.05, p < 0.01, and p < 0.001, respectively.