Mouse Models

All animal procedures were performed in accordance with the Principles of Laboratory Animal Care and Guidelines approved by the Dutch Animal Ethical Committee in full accordance with European legislation. As required by Dutch law, formal permission to generate and use genetically modified animals was obtained from the responsible local and national authorities. A total of 85 mice, males and females, were used for this study. Animals were housed in individual ventilated cages under specific pathogen free conditions and maintained in a controlled environment (20–22 ˚C, 12 h light: 12 h dark cycle). They were given ad libitum access to food, maintained on either AIN93G synthetic pellets (Research Diet Services B.V.; gross energy content 4.9 kcal/g dry mass, digestible energy 3.97 kcal/g) or standard chow diet and water.

The generation of Ercc1Δ/− mutants (carrying a genetic mutation in which one of the alleles is removed, “knock-out”, and the other allele has a C-terminally truncation, “delta”, leading to the production of a truncated version of the Ercc1 protein (Ercc1Δ/−)) and their wild-type Ercc1+/+ littermates (WT) has been described previously [13]. These mice, all in a F1 hybrid FVB/C57BL/6 J background, were studied at 6, 12 and 24 weeks of age. Typical unfavorable characteristics, such as blindness in the FVB background or deafness in the C57BL/6 J background, do not occur in this hybrid background. Mice with cardiomyocyte-restricted deletion of Ercc1 (αMHC-Ercc1c/−) were generated using the Cre-loxP technology as previously described [14]. Mice harboring two conditional alleles of Ercc1 were crossed with hemizygous mice expressing Cre-recombinase under the control of the α-myosin heavy chain (αMHC-Cre) promotor [17]. αMHC-Ercc1c/− mice and their control littermates (Control) harboring the same F1 hybrid FVB/C57BL/6 J background, were studied at 8 and 16 weeks of age.

In Vivo Micro-CT Imaging of LV Function

Mice were imaged with contrast enhanced Quantum FX Micro-computed Tomography (micro-CT) (Perkin Elmer Inc., Akron, Ohio, USA) for anatomical reference and to assess cardiac morphology and function [18].

Mice were anaesthetized (1.5–2.5% isoflurane, O2 1 L/min) and depilated to minimize the interference of fur on the fluorescent signal during FMT imaging. Prior to micro-CT imaging (Perkin Elmer Inc.), mice were injected in the tail vein with 125 µL per 25 g body weight of the iodinated contrast agent eXIA160 (10 mg/ml, Binitio Biomedical Inc., Ottawa, Canada), positioned in the multimodal imaging cassette and restrained to prevent movement during imaging. Mice were scanned using intrinsic cardiac respiratory gating to reduce artifacts caused by breathing or cardiac motion. After micro-CT imaging, mice remained under anesthesia and the cassette was transferred to the FMT 2500 fluorescent tomography in vivo imaging system (Perkin Elmer Inc.). Indexes of LV myocardial mass (LVMM) and LV function (LV end-diastolic volume, LV end-systolic volume and LV stroke volume) were measured from the 3D micro-CT images using the software ANALYZE ® 12.0 (AnalyzeDirect Inc., Overland Parks, KS, USA). Subsequently, stroke volume was calculated as LV-end-diastolic volume - LV end-systolic volume, while LV ejection fraction was calculated using this formula: ((LV end-diastolic volume-LV end-systolic volume)/LV end-diastolic volume)*100%.

In Vivo FMT Imaging of Myocardial Apoptosis

The near-infrared fluorescence (NIRF) probe Annexin-Vivo750™ (Perkin Elmer Inc.) was used to image myocardial apoptosis. Therefore, the FMT 2500 fluorescence tomography in vivo imaging system (Perkin Elmer Inc.) was used. Two hours before imaging, mice were injected intravenously with Annexin-Vivo 750™ (according to manufacturer’s instructions). After finishing micro-CT imaging, the cassette was transferred to the FMT 2500 fluorescent tomography in vivo imaging system (Perkin Elmer Inc.). FMT imaging was performed using 750 and 770–800 nm excitation and emission wavelengths, respectively. The multimodal imaging cassette facilitates the co-registration of FMT and micro-CT data through fiducial landmarks. FMT and micro-CT data were merged using the TrueQuant software (Perkin Elmer Inc.) and in vivo cardiac fluorescence was quantified as the amount of pmol divided by heart weight (g).

Ex Vivo Fluorescent Imaging of Excised Hearts

Immediately after in vivo micro-CT and FMT imaging, mice were euthanized using an overdose of inhalant anesthetic isoflurane. Hearts were excised, weighed, immersion fixed in formalin for 1 day and assessed for ex vivo tissue epifluorescence using the Odyssey® CLx imaging system (LI-COR® Biosciences, Lincoln, Nebraska, USA). Ex vivo cardiac fluorescence was quantified as counts per mm2.

Immunohistochemical Analysis

After formalin fixation, the heart was transversally sectioned into two parts and stored overnight in 70% ethanol. Subsequently, hearts were embedded in paraffin, sectioned at 5 µm, and mounted on Superfrost Plus slides. Cross-sections of the whole heart were stained for TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling, ApopTag In Situ Apoptosis Kit (Oncor)) to determine the amount of apoptotic cells using standard procedure. TUNEL staining was quantified by counting TUNEL positive (dark brown) cells in qualitatively suitable sections from the heart. This number was corrected for the size of the quantified area by dividing the total fragmentation count by the surface of the measured area (cells/mm2).

Statistical Analysis

Data are expressed as mean ± SEM. Survival analysis was performed with the Mantel-Cox test using GraphPad Prism 10.1.0. Differences between groups were evaluated by two-way ANOVA, followed by Student–Newman–Keuls (SNK) post-hoc testing using SigmaPlot 11.0. P < 0.05 (two-tailed) was considered significant.