A modification of retrograde Langendorff‐perfusion to increase the yield of Ca2+-tolerant murine atrial cardiomyocytes isolated from remodelled, fibrotic atria

Figures

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  FIGURE 1. Overview of study design. Isolations of aCM were performed according to standard protocol (STD) and with additional ventricular ligature (MOD) (exemplary images and schematic views of heart perfusion shown) for each 3 animals of CTR and TG group. aCM suspension was examined both at 0 and 1 mmol/L Ca2+ after trypan blue staining of each 3 technical replicates. 10 μL of stained aCM suspension were placed on a slide and a microscopic image was acquired of the whole aCM suspension drop (stitched exemplary microscopic image shown). aCM were counted and classified depending on morphological intactness and trypan blue uptake (exemplary aCM shown in table). Only aCM with intact morphology and without trypan blue uptake were considered eligible for functional single-cell assessment (green box). aCM: atrial cardiomyocytes, Ca2+: calcium, CTR: control group, TG: transgenic group (CREM-IbΔC-X mice), STD: standard isolation protocol, MOD: modified isolation protocol.

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  FIGURE 2. Extensive atrial remodelling in mice with CREM-IbΔC-X overexpression (A) Exemplary cannulated hearts. (B) 2D-area of perfused hearts: severe dilatation in TG hearts. (C) Body weight. (D) Relative atrial mass to body weight: raised in TG hearts. (E) Exemplary Masson-Trichrome-stained hearts with 5× magnified atrial wall sections, the arrow marks an atrial thrombus as often observed in TG atria. (F) Atrial fibrosis determined by proportion of blue stained tissue: enhanced atrial fibrosis in TG atria. (12 weeks, n = 6 per group, except for 2D-area: n = 5 per group, t-test *p < 0.05). CTR: control group, TG: transgenic group (CREM-IbΔC-X mice).

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  FIGURE 3. Reduced aCM yield with established Langendorff-perfusion protocol from remodelled atria (A) Schematic view of a Langendorff-perfusion-setup with components: 1) roll pump, 2) standard counterflow heat exchanger, 3) cannula, 4) perfused heart in 5) heated bath, 6) three-way stopcock for solution exchange, 7) heating circulator, 8) waste. (B) Exemplary picture of the used setup. (C) Total aCM yield per 10 µL: lower total aCM-extraction from TG vs. CTR atria. (D) Count of intact aCM per 10 µL: lower at 0 mmol/L Ca2+ in TG, also a tendency to lower yield of intact aCM from TG atria at 1 mmol/L Ca2+. (n = 3 vs. 3 per genotype, 3 technical replicates per isolation (raw data points, colour-coded per isolation), statistics on mean per isolation, t-test *p < 0.05 vs. CTR) aCM: atrial cardiomyocytes, CTR: control group, TG: transgenic group (CREM-IbΔC-X mice).

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  FIGURE 4. Ventricular ligature increases yield of Ca2+-tolerant aCM from remodelled atria (A) heart without (STD) and with ventricular ligature (MOD) visualising the modification of the established protocol. (B) Schematic hypothesis of changed myocardial perfusion by the ligature. (C) Total aCM/10 µL and (D) intact aCM/10 µL in absence of Ca2+(0 mmol/L): unchanged by MOD protocol in CTR hearts vs. STD, while there were tendencies to higher aCM yields with MOD protocol from TG atria. (E) Total aCM/10 μL at 1 mmol/L Ca2+: significantly higher total aCM extraction from TG atria with MOD vs. STD (p < 0.05). (F) Intact aCM/10 μL at 1 mmol/L Ca2+: tendency to higher counts of intact aCM both from CTR and TG atria with MOD vs. STD (n.s.). (n = 3 vs. 3, 3 technical replicates per isolation (raw data points, colour-coded per isolation), data for STD are identical to those shown in Figures 3C,D statistics on mean per isolation, t-test *p < 0.05 vs. STD). aCM: atrial cardiomyocytes, CTR: control group, TG: transgenic group (CREM-IbΔC-X mice), STD: standard isolation protocol, MOD: modified isolation protocol.

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  FIGURE 5. aCM respond well to electrical field stimulation (A) representative images of morphologically intact aCM isolated with the modified protocol. (B) Proportion of morphologically intact aCM analysed under the indicated conditions that responded reliably with field stimulation-induced Ca2+ transients without spontaneous activity. (C) Representative traces of Ca2+ transients, recorded under basal conditions (top) and during stimulation with 1 μmol/L isoproterenol (bottom) in CTR (left) and TG (right) aCM isolated with the modified protocol. (n = 6 isolations per genotype, 443 aCM in total, 12–34 aCM per data point shown in (B)). aCM: atrial cardiomyocytes, BASAL: basal conditions, CTR: control group, ISO: isoproterenol stimulation, RU: relative units, TG: transgenic group (CREM-IbΔC-X mice).