Rejuvenating the Aging Heart by Enhancing the Expression of the Cisd2 Prolongevity Gene

Figure 1. Schematic illustration of the inducible and cardiac-specific overexpression of Cisd2 (Cisd2 icOE) in transgenic mice. (A) The strategy used for cardiac-specific and tamoxifen (TAM)-inducible overexpression of Cisd2 in the mice. The R26-Cisd2KI/+ mice were crossed with mice carrying the cardiac-specific expression of tamoxifen-inducible Cre recombinase (αMHC-MerCreMer). (i) In the absence of tamoxifen, the MerCreMer (MCM) is sequestered by heat shock protein 90 (HSP90) and retained in the cytoplasm. (ii) Under the presence of tamoxifen, the MCM complex is disassociated from HSP90 and is then translocated into the nucleus to carry out the Cre/loxP-mediated deletion of the STOP cassette; αMHC, α-myosin heavy chain promoter. (B) The flow chart of tamoxifen treatment. The cardiac functions were monitored at 3 and 18 months of age before tamoxifen injection. At 18 months of age, both the WT control (R26-Cisd2KI/+) and the Cisd2 icOE (R26-Cisd2KI/+; αMHC-MCM) mice had tamoxifen administered by intraperitoneal injection (40 mg/kg body weight on three consecutive days). Cardiac function analysis was performed at 26 months of age, namely 8 months after tamoxifen injection. (C,D) Western…

Figure 2. Cardiac-specific overexpression of Cisd2 at a late-life stage rescues age-related electromechanical dysfunction of the aging heart. (A) Representative echocardiographs obtained from the WT control (R26-Cisd2KI/+) mice and the Cisd2 icOE (R26-Cisd2KI/+; αMHC-MCM) mice (before 18 months of age) and after tamoxifen injection for 8 months (26 months). White bar: 0.1 s. Echocardiographs of the 3-mo WT mice were used as the control. (B,C) Left ventricular ejection fraction (EF) and end-diastolic diameter were measured and quantified by echocardiography. Left ventricular EF decreased in the WT (R26; Cisd2KI/+) mice during aging. However, in the Cisd2 icOE mice, their EF had increased at 26 months of age after induction of Cisd2 overexpression for 8 months compared with that at 18 months. In the WT mice, the LVEDD significantly increased from the age of 18 months to the age of 26 months. However, in the Cisd2 icOE mice, the LVEDD did not increase at 26 months of age compared with 18 months of age. (D) Representative waterfall plots and ECG tracings recorded following anesthesia of the mice. Atrioventricular (AV) block with irregular and progressive prolongation of the PR interval (E), a…

Figure 3. Cardiac-specific overexpression of Cisd2 at a late-life stage rescues the aging hearts of mice from age-related cardiac fibrosis and intercalated disc (ICD) defects. (A) Age-related cardiac fibrosis can be seen to be ameliorated by enhancing the expression level of Cisd2 in the aging heart. Representative immunofluorescence images of heart sections from the three groups of mice (3-mo WT, 26-mo WT, and 26-mo Cisd2 icOE) stained with antibodies against collagen I (green) and wheat germ agglutinin (red) to identify the cell membrane, with rhodamine phalloidin—to identify actin (purple), and with DAPI—to identify the cardiomyocytic nucleus. Age-related cardiac fibrosis is obvious in the 26-mo WT mice (A2). However, overexpression of cardiac Cisd2 appears to reduce cardiac fibrosis (A3). (B,C) Overexpression of Cisd2 reverses the age-related defects affecting the ICD in the aging heart. Representative immunofluorescence images of heart sections from the three groups of mice (3-mo WT, 26-mo WT, and 26-mo Cisd2 icOE) stained with antibodies against Cx43 (green) was used to localize gap junctions, against pan-cadherin (red)—to localize the ICDs, and against α-actinin (purple)—to stain…

Figure 4. Cardiac-specific overexpression of Cisd2 at late-life stage in Cisd2 icOE mice reverses the cardiac transcriptomic profile changing it from an old-age pattern to a younger pattern. (A) Principal component analysis (PCA) of the RNA-seq data from the three groups of mice, namely 3-mo WT, 26-mo WT, and 26-mo Cisd2 icOE. (B) Volcano plots representing the transcriptomic changes in the 26-mo WT vs. 3-mo WT mice and in 26-mo WT vs. 26-mo Cisd2 icOE mice. The horizontal lines show the 25% false discovery rate (FDR) or the p < 0.05 threshold. Blue dots indicate the downregulated genes and red dots indicate the upregulated genes. (C) Heatmap showing the 97 differently expressed genes (DEGs) that are reverted by the cardiac-specific overexpression of Cisd2 in the Cisd2 icOE mice. (D) Analysis of the Cisd2-reverted DEGs by the GO Biological Processes. (E) Heatmap showing the DEGs involved in Golgi and protein transport, mitochondrial (Mt.) energy metabolism and redox, and ICDs and ECM. ICD, intercalated disc; ECM, extracellular matrix.

Figure 5. Analyses of the biological functions and pathways of the DEGs reverted by cardiac-specific Cisd2 overexpression. (A) Ingenuity pathway analysis (IPA) of the DEGs. (B) Heatmaps representing the Cisd2-reverted DEGs (26-mo Cisd2 icOE vs. 26-mo WT mice, p < 0.05) related to the sirtuin, autophagy, and senescence pathways. (C) The diagram illustrates the relationships between the SIRT family genes and their related biological functions and the Cisd2-reverted genes. The significant genes reverted by Cisd2 are highlighted with blue color (p < 0.05). (D) The upstream regulators obtained by IPA upstream regulator analysis. The figure was created with BioRender.com.

Figure 6. Cardiac-specific overexpression of Cisd2 at the late-life stage rejuvenates the aging heart. (A) In the WT mice at an old age, a decrease in cardiac Cisd2 results in mitochondrial dysfunction and metabolic reprogramming, as well as in the increase in ROS production, dysregulated proteostasis, and disrupted cytosolic Ca2+ homeostasis. Consequently, these defects lead to cellular damage to cardiomyocytes, as well as degeneration of the ICDs, an increase in interstitial fibrosis, and remodeling of the extracellular matrix together with dysregulation of cardiac electromechanical functions. As a result, these functional and structural alterations, which are accompanied by chronic inflammation, bring about cardiac aging. (B) In the Cisd2 icOE mice at an old age, cardiac-specific overexpression of Cisd2 at the late-life stage appears to rejuvenate the aging heart by reversing the age-related structural damage and the functional decline of the heart. ECM, extracellular matrix; ETC, electron transport chain; ICD, intercalated disc; Mt., mitochondrial; TCA, tricarboxylic acid cycle. The figure was created with BioRender.com.