Cisd2 delays atrial aging via a modulation of calcium homeostasis that mitigates atrial myopathy

Abstract

Age-associated atrial myopathy results in structural remodeling and a disturbance of atrial conductance. Atrial myopathy often precedes atrial fibrillation (AF) and can facilitate AF progression. However, the molecular mechanism linking aging to atrial deterioration remains elusive. CDGSH iron-sulfur domain-containing protein 2 (CISD2) is a mammalian pro-longevity gene. We used Cisd2 knockout (Cisd2KO) and Cisd2 transgenic (Cisd2TG) mice to investigate pathophysiological mechanisms underlying age-related atrial myopathy. Four findings are pinpointed. Firstly, in both humans and mice, the level of atrial CISD2 declines during natural aging; this correlates with age-associated damage, namely degeneration of intercalated discs, mitochondria, sarcoplasmic reticulum (SR) and myofibrils. Secondly, in Cisd2KO and naturally aged wild-type mice, Cisd2 deficiency causes atrial electrical dysfunction and structural deterioration; conversely, sustained Cisd2 levels protect Cisd2TG mice against age-related atrial myopathy. Thirdly, Cisd2 plays a vital role in maintaining Ca²⁺ homeostasis in atrial cardiomyocytes. Cisd2 deficiency disrupts Ca²⁺ regulation, leading to elevated cytosolic Ca²⁺, reduced SR Ca²⁺, impaired store-operated calcium entry, and mitochondrial Ca²⁺ overload; these compromise mitochondrial function and attenuate antioxidant capability. Finally, transcriptomic analysis reveals that Cisd2 protects the atrium from metabolic reprogramming and preserves into old age a transcriptomic profile resembling a youthful pattern, thereby safeguarding the atrium from age-related injury. This study highlights Cisd2's crucial role in preventing atrial aging and underscores the therapeutic potential of targeting Cisd2 when combating age-associated atrial dysfunction, which may lead to the development of strategies for improving cardiac health in aging populations.

Keywords: Aging; Atrial fibrillation; Atrial myopathy; Calcium homeostasis; Cisd2.

Fig. 1

Age-dependent decrease of atrial CISD2 protein level is associated with PR interval prolongation and atrial arrhythmias in humans. In each decade of age, from 20’s until the 90’s, the average of PR intervals (A), the coefficient of variance of the PR intervals (B), and the prevalence of atrial fibrillation or flutter (C) were found to significantly increase during aging in both male and female patients. (D) and (E), Western blot analyses and quantification of CISD2 in human atrial specimens revealed that there was a decrease of atrial CISD2 level and this was significantly associated with aging. (F) Representative IF images of human atrial sections stained with antibodies against Cx43 (green) to localize gap junctions, against pan-cadherin (red) to localize the intercalated discs, and against α-actinin (purple) to stain muscle fibers in male and female patients with high and low CISD2 expression. The sections were also strained with Hoechst (blue) to identify nuclei. (G) Colocalization coefficients of gap junctions (Cx43) and intercalated discs (pan-cadherin) were analyzed using Pearson’s correlation. (H) Representative IF images of human atrial sections stained with antibodies against desmoplakin (green) to localize desmosome, against pan-cadherin (red) to localize the intercalated discs, and against α-actinin (purple) to stain muscle fibers. The sections were also strained with Hoechst (blue) to identify nuclei. (I) Colocalization coefficients of desmosome (desmoplakin) and intercalated discs (pan-cadherin) were analyzed using Pearson’s correlation. The corresponding lower power photomicrographs of (F) and (H) are presented in Supplementary Figure S2A and Supplementary Figure S2B

Fig. 2

Cisd2 deficiency in the atrium results in functional and structural deterioration, while a high level of Cisd2 ameliorates age-associated atrial myopathy. (A) and (B), Western blot analyses and quantification of atrial Cisd2 protein levels. (C)-(F), Representative waterfall plots and ECG tracings obtained from 3-mo WT (C), 3-mo Cisd2KO (D), 24-mo WT (E), and 24-mo Cisd2TG (F) mice. Representative atrial dysrhythmic ECGs, namely irregular and prolonged PR intervals and AV block, were found in the 3-mo Cisd2KO and 24-mo WT mice. (G) and (H), Quantification of the average and the CV (coefficient of variance) of ECG PR intervals in various mouse groups. (I) and (K), Representative IF images of atrial sections stained with antibodies against Cx43 (green) to localize gap junctions in (I) and against desmoplakin (green) to localize desmosome in (K). The sections were also stained with antibodies against pan-cadherin (red) to localize the ICDs, and with antibodies against α-actinin (purple) to stain muscle fibers. In addition, the sections were strained with Hoechst (blue) to identify nuclei. (J) and (L), Colocalization coefficient of gap junctions (Cx43) (J) and desmosomes (desmoplakin) (L) with ICDs (pan-cadherin) was analyzed using Pearson’s correlation. The data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA with Bonferroni multiple comparison test. The corresponding lower power photomicrographs of (I) and (K) are presented in Supplementary Figure S4A and Supplementary Figure S4B

Fig. 3

CISD2 protects the atrial ultrastructure against age-associated injuries in humans and mice. (A) Representative TEM of the right atrium sampled from a patient with high CISD2. Relatively normal ultrastructures are found, namely intact mitochondria, gap junctions, SR and cardiac myofibrils. (B) Schematic presentation of the ultrastructure of the right atrium shown in (A). (C) Representative TEM of the right atrium sampled from a patient with low CISD2. Mitochondria degeneration, lipofuscin accumulation, and necrotic debris from degenerating myofibrils and organelles can be seen and easily identified. (D) Schematic presentation of the ultrastructure of right atrium shown in (C). (E) Representative TEM for the right atrium of a 24-mo WT mouse. Degeneration of mitochondria and myofibrils can be easily observed. The blurring of the Z line and disarrangement of the ICDs resulting in expanded intercellular spaces can also be detected. (F) Schematic presentation of the ultrastructure of the right atrium shown in (E). (G) Representative TEM of the right atrium of a 24-mo Cisd2TG mouse. The high level of Cisd2 preserves the ultrastructure of organelles and ICDs in Cisd2TG mice. (H) Schematic presentation of the ultrastructure of the right atrium shown in (G). Abbreviations: Cap, capillary; Cm, cardiac myofibril; Cmd, cardiac myofibril degeneration; D, desmosome; EIS, expanded intercellular space; F, fibrosis; FA, fascia adherens; GJ, gap junction; d. ICD; degenerated intercalated disk (ICD f., ICD fragmentation); Lf, lipofuscin; IS, intercellular space; M, mitochondria; MD, mitochondrial degeneration; ZL, Z line; ZLb: Z line breakdown

Fig. 4

Cisd2 deficiency disrupts intracellular Ca²⁺ homeostasis, and causes mitochondrial Ca²⁺ overload and dysfunction in HL-1 atrial cardiomyocytes. (A) Western blot analysis of Cisd2 in HL-1 cells carrying a WT, Cisd2KO, and Cisd2RE genetic background. (B) Schematic illustrating intracellular Ca²⁺ regulation via the SR Ca²⁺pump (SERCA) and a Ca²⁺channel (RyR2), as well as the store-operated calcium entry (SOCE)-related channel (Orai1) and various regulators (TRPC1, STIM1 and STIM2) in cardiomyocytes. Cisd2 maintains intracellular Ca²⁺ homeostasis via the modulation of SERCA2 activity. Nifedipine is a Ca²⁺ channel blocker that inhibits voltage-gated Ca²⁺ channels (VGCCs) leading to a decrease in Ca²⁺ influx from extracellular area to the cytosol. SKF-96,365 is a TRPC1 inhibitor that is able to reduce Ca²⁺ influx via an inhibition of SOCE. (C) The levels of cytosol Ca²⁺ in single HL-1 cells were measured by fluorescence microscopy using Fura-2/AM staining. After measuring the basal level of cytosol Ca²⁺ (first 50 s), thapsigargin (Tag) was added to release Ca²⁺ from the SR. (D) Quantification of the basal cytosol Ca²⁺ levels in the HL-1 cells. (E) Quantification of peak Ca²⁺ release from the SR (maximum minus basal) after Tag treatment as in (C). (F) Quantification of the SOCE levels in the HL-1 cells. (G) and (H), Representative confocal imaging and quantification of STIM1 puncta formation in response to SR Ca²⁺ depletion. (I) Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added to release Ca²⁺ from the mitochondria. (J) Quantification of peak Ca²⁺ release from mitochondria (maximum minus basal) after CCCP treatment. (K) and (L), Oxygen consumption rates (OCR) of the HL-1 cells. The indicated chemicals (OA, oligomycin A; FCCP, carbonilcyanide p-triflouromethoxyphenylhydrazone; Rot/AA, rotenone/antimycin A), were added sequentially to determine the ATP-coupled respiration rate, the maximal respiration rate (Max), and the non-mitochondrial respiration rate, respectively, using a Seahorse XF^e 24 analyzer. (M) The ROS levels of WT, Cisd2KO and Cisd2RE HL-1 cells after H₂O₂ (0, 10, and 100 µM) treatment were measured by DCF-DA. In (D)-(F) and (J), the data are presented as mean ± SEM of 60 to 76 cells in two independent experiments. In (H), (L) and (M), the data are presented as mean ± SD from at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA with Bonferroni multiple comparison test

Fig. 5

A persistently high level of Cisd2 preserves a younger transcriptomic profile in the atrium of Cisd2TG mice during old age. (A) PCA analysis of atrial aging-related DEGs (25-mo WT vs. 3-mo WT; FDR < 0.05) in the atrial tissues of 3-mo WT, 25-mo WT and 26-mo Cisd2TG mice. The PCA was performed using MetaboAnalyst v6.0 (https://www.metaboanalyst.ca/). (B) The biological processes of GO annotation of the Cisd2TG-reverted transcriptome changes (219 DEGs). The grouping of the GO annotation was carried out by STRING v11.5 (https://string-db.org/). FDR < 0.05 was used for selection. (C) The grouping of the KEGG pathways of the Cisd2TG–reverted 219 DEGs was carried out using KOBAS-i (http://bioinfo.org/kobas). A p < 0.05 was used for pathway selection. (D) Canonical pathway analysis by IPA of the Cisd2TG-reverted 219 DEGs. A p < 0.05 was used for pathway selection. (E) The mRNA levels of the integrin-FAK pathway-related DEGs. (F) The mRNA levels of the JAK-STAT pathway-related DEGs. (G) The mRNA levels of the GPCR-RAS pathway-related DEGs. (H) The mRNA levels of the PI3K-AKT-NF-κB pathway-related DEGs. (I) The mRNA levels of the AGEs-RAGE pathway-related DEGs. (J) A graphic summary of the DEGs and pathways associated with natural aging in WT mice and long-lived Cisd2TG mice. In the aged WT mice, various cardiac aging-related pathways have an impact on the atrium during natural aging; these, include the GPCR-RAS, Integrin-FAK, JAK, PI3K-AKT-NF-κB and AGEs-RAGE signaling pathways. In the GPCR-RAS pathway, the expression of the upstream regulators of RAS, including Cxcl16, Grk2, Grk5, and Gng11, were increased in the atrium of the aged WT mice. Moreover, the downstream targets of Ras signaling, including Stk4 and Ets1, exhibit increased expression in the atrium of aged WT mice. Stk4 and Ets1 are involved in cell cycle arrest and cell death,, respectively, as well as cellular senescence. In the JAK-PI3K-AKT-NF-κB pathway, key factors, such as Jak1, Jak3, Akt3 and Nfkb2, exhibit enhanced expression in the atrium of aged WT mice. The upregulation of Akt3 and Nfkb2 is able to promote cardiac hypertrophy, inflammation and fibrosis. In the integrin-FAK pathway, the upstream regulators of FAK, including Sparcl1, Itga1 and Itga6, exhibit increased expression in the atrium of aged WT mice. Activated FAK can modulate downstream signaling regulators, including Ras and PI3K, thereby promoting cardiac hypertrophy, remodeling and fibrosis. In the AGEs-RAGE pathway, dysregulation of the downstream targets of RAGE, including Plcd3 and PKCδ, is observed in the atrium of aged WT mice. Elevated PKCδ can increase ROS levels and promote PI3K signaling. These dysregulated signaling pathways collectively contribute to the aging-related pathological alterations in the atrium of aged WT mice. Cisd2 overexpression reverses these dysregulated gene expression profiles, thereby ameliorating the pathological changes in the atrium of old Cisd2TG mice

Fig. 6

A variety of metabolic pathways, namely amino acid metabolism, lipid metabolism, NRF2-mediated oxidative stress and mitochondrial UPR, are activated in the atrium of Cisd2KO mice. (A) PCA analysis of Cisd2KO-related DEGs (3-mo Cisd2KO vs. 3-mo WT; FDR < 0.05). The PCA was performed by MetaboAnalyst v6.0 (https://www.metaboanalyst.ca/). (B) The grouping of the KEGG pathways of Cisd2KO-related DEGs was carried out using KOBAS-i (http://bioinfo.org/kobas). A p < 0.05 was used for pathway selection. (C) Canonical pathway analysis by IPA of the Cisd2KO-related transcriptome changes. A p < 0.05 was used for pathway selection. (D) A heatmap illustrating that Cisd2 deficiency affects the expression of a panel of DEGs (Cisd2KO vs. WT, FDR < 0.05) that are involved in the following: (i) amino acid metabolism, (ii) NAD⁺ and Sirtuin-PPARα, (iii) NRF2-mediated oxidative stress response, and (iv) mitochondrial unfolded protein response (mtUPR). (E) A graphic summary of the DEGs and pathways associated with Cisd2 deficiency in the atrium of Cisd2KO mice. In the Cisd2KO mice, various cardiac aging-related biological processes and pathways have an impact on the atrium during premature aging. (i) Amino acid metabolism: the expression of several enzymes involved in valine and isoleucine degradation, including Dbt, Acadsb, Hsd17b10, Aldh6a1 and Aox1, is significantly enhanced in Cisd2KO mice. Additionally, Aldh6a1, an enzyme involved in alanine metabolism, is significantly increased in the Cisd2KO atrium. (ii) The NAD⁺ and Sirtuin-PPARα pathway: Nampt, the enzyme converting nicotinamide (NAM) to nicotinamide mononucleotide (NMN), is significantly increased in Cisd2KO atrium, leading to increases in NAD⁺ production and Sirtuin 1 (Sirt1) activity. Subsequently, Sirt1 modulates its downstream targets by inhibiting Hif1α; this in turn regulates the TCA cycle. In addition, Sirt1 activates PPARα and its downstream targets (Abca1, Acox1 and Gpd1) expression, thereby influencing lipid metabolism in the atrium. (iii) The NRF2-mediated oxidative stress response: Cisd2 deficiency leads to increase of ROS levels and activates NRF2 and its downstream target gene expression, namely Aox1, Dnajc7 and Fom1. The enhanced Aox1 and Fom1 are able to further increase ROS levels, thus establishing a vicious cycle of oxidative stress in the atrium of Cisd2KO mice. (iv) Mitochondrial dysfunction and mtUPR: Cisd2 deficiency causes mitochondrial dysfunction and enhances the expression levels of mtUPR-related chaperones, namely Dnajc7 (Hsp40) and Hspa9 (mtHsp70), in the atrium. Taken together, these changes would seem to contribute to the premature aging phenotype that affects the atrium of Cisd2KO mice