Histological analyses

5.3.4.4.1 Evaluation of H&E-stained embryonic, P1 and adult hearts

To assess overall cardiac morphology and tissue composition, light microscopy images of H&E-stained hearts (see Chapter 5.3.4.2.1) were taken using the Biozero BZ-8100 microscope (Keyence). For 10.5 dpc embryonic hearts 5-fold optical magnification, for 13.5 dpc embryonic hearts 4-fold optical magnification and for P1 hearts 2-fold optical magnification was applied. Hearts of 11 week old adult mice were imaged with 2-fold optical magnification. Due to the size of the adult heart, however, four overlapping picture excerpts were taken, which were subsequently merged into a single image using the BZ image analysis application software (Keyence). Pictures of at least two non-adjacent longitudinal (embryonic and P1 hearts) or cross (adult) H&E-stained heart sections were imaged.

METHODS

5.3.4.4.2 Quantification of fibrosis in adult hearts

Light microscopy images of Sirius red-stained hearts (see Chapter 5.3.4.2.2) from 11 week old adult mice were taken using the Biozero BZ-8100 microscope (Keyence). By applying 5-fold optical magnification, 6 random fields of the left ventricular (LV) myocardium, including free wall and interventricular septum (IVS), were imaged, thereby covering the entire LV tissue. Two non-adjacent Sirius red-stained cross-sections per heart were used.

The percentage of interstitial LV fibrotic tissue was quantified using the color-threshold-plugin of the ImageJ software, which measures the red stained area in relation to the total LV myocardial area. Fibrosis of all 12 images was averaged to give a mean estimate of fibrotic tissue for each heart. Perivascular fibrosis was excluded and was cut-out in the images before analysis. Imaging measurements as well as analyses took place in a blinded fashion, such that investigators were unaware of mouse genotypes or treatment groups.

5.3.4.4.3 Measurement of cardiomyocyte cross sectional area in P1 and adult hearts

To measure cardiomyocyte cross sectional area (CSA), WGA-stained P1 longitudinal or 11 week old adult heart cross-sections were used (see Chapter 5.3.4.3.1). For P1, pictures were taken with the confocal laser scanning microscope TSC SPE (Leica Microsystems) applying 120-fold optical magnification. For adult hearts, pictures were taken with the Biozero BZ-8100 fluorescence microscope (Keyence) applying 20-fold optical magnification. For P1 as well as adult hearts, 15 random fields of the LV myocardium, including free wall and IVS, were imaged and two non-adjacent WGA-stained sections per heart were used. At least 200 cardiomyocytes per heart were measured using the area measurement tool of the Biozero BZ image analysis application software (Keyence). Only CSA of cardiomyocytes which were cut along their transverse axis and matched the following selection criteria was measured: visible central nucleus, cell shape close to circular, clear borders of cell discernible and visible cytoplasm. CSA of all cardiomyocytes analyzed were averaged to give a mean estimate of cardiomyocyte CSA for each heart. All measurements as well as analyses took place in a blinded fashion, such that investigators were unaware of mouse genotypes or treatment groups.

5.3.4.4.4 Quantification of apoptosis rates in embryonic 13.5 dpc and P1 hearts

To quantify the number of apoptotic cells, TUNEL-stained 13.5 dpc or P1 hearts were analyzed (see Chapter 5.3.4.3.2). Two longitudinal non-adjacent sections per heart were imaged and pictures of 6 random fields per heart section (entire LV and RV myocardium for 13.5 dpc, LV myocardium for P1) were taken using the Biozero BZ-8100 fluorescence microscope (Keyence) with 15-fold optical magnification. Cells which exhibited nuclear colocalization of TUNEL and DAPI staining were considered apoptotic. Nuclei with positive

METHODS

TUNEL staining as well as the total number of DAPI-stained nuclei were manually counted using the cell-counter-plugin of the ImageJ software. A total number of approximately 7,000 cells per embryonic heart and 15,000 cells per P1 heart was analyzed. Apoptosis rates (given in percent) were calculated by relating the number of TUNEL-positive nuclei to the total number of nuclei as mean from all images per heart. All measurements as well as analyses took place in a blinded fashion, such that investigators were unaware of mouse genotypes or treatment groups.

5.3.4.4.5 Quantification of proliferation rates in neonatal hearts

To quantify the number of proliferating cells in P1 hearts, KI67-stained longitudinal sections (see Chapter 5.3.4.3.3) were imaged with 40-fold optical magnification using the Biozero BZ-8100 fluorescence microscope (Keyence). Pictures of 10 random fields of one heart section were taken within the LV myocardium, including the free wall and IVS. Cells that exhibited nuclear colocalization of KI67 and DAPI were considered as cycling. KI67-positive nuclei and the total number of DAPI-stained nuclei were manually quantified using the cell-counter-plugin of the ImageJ software. A total number of approximately 5,000 cells per heart was evaluated with respect to their resting or proliferating stage. The proliferation rate per heart was calculated as the percentage of KI67-positive cells related to the total number of DAPI-stained nuclei. All measurements as well as analyses took place in a blinded fashion, such that investigators were unaware of mouse genotypes or treatment groups.

5.3.4.4.6 Quantification of proliferation rates in embryonic 13.5 dpc hearts

To quantify the number of proliferating cells in embryonic 13.5 dpc hearts, p-HH3-stained longitudinal sections (see Chapter 5.3.4.3.4) were imaged with 30-fold optical magnification using the confocal laser scanning microscope TSC SPE (Leica Microsystems). Pictures of 6 to 11 random fields of two non-adjacent heart sections were taken within the LV and RV myocardium. Cells which exhibited nuclear colocalization of p-HH3 and TO-PRO®-3 were considered as cycling. P-HH3-positive nuclei and total number of TO-PRO®-3-stained nuclei was manually quantified using the cell-counter-plugin of the ImageJ software. The number of cells in mitosis was taken as the total p-HH3-positive cells over the total TO-PRO®-3-labeled nuclei of each cell population analyzed. Within cHccs^+/- hearts, CYT c staining was used to differentiate between the healthy and Hccs-deficient myocardial cell population. The proliferation rate of healthy myocardial cells was calculated as the percentage of p-HH3-positive cells related to the total number of TO-PRO®-3-stained nuclei within CYT c-p-HH3-positive cells. The proliferation rate of Hccs-deficient myocardial cells was calculated as the percentage of p-HH3-positive cells related to the total number of TO-PRO®-3-stained nuclei within CYT c-negative cells. In Hccs^+/+ hearts, all cardiomyocytes were CYT c-positive and

METHODS

the proliferation rate per heart was calculated as the percentage of p-HH3-positive cells related to the total number of TO-PRO®-3-stained nuclei. A total number of approximately 5,000 cells per heart was evaluated with respect to their resting or proliferating stage. All measurements as well as analyses took place in a blinded fashion, such that investigators were unaware of mouse genotypes or treatment groups.