300-µm rat Living Myocardial Slices (LMS) cut with 7000smz-2 vibrotome

Abstract

(from: CaMKII inhibition reduces arrhythmogenic Ca2+ events in subendocardial cryoinjured rat living myocardial slices, Dries et al, 2021)

Spontaneous Ca2+ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca2+ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca2+ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca2+ transients to peak after injury. In ENDO LMS, the Ca2+ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation–contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies.

Method

Preparation of LMSs

Preparation of LMSs was done according to a protocol previously established by our laboratory (Watson et al., 2017). The rat heart was immediately removed and placed in warm (37°C) and later ice-cold (4°C), heparinized (2.4 IU/ml), normal Tyrode’s solution (in mmol/liter: 140 NaCl, 6 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4 with NaOH) containing 30 mM 2,3-butanedione monoxime (BDM) to remove all excessive blood from the heart. The LV was isolated, opened flat, and mounted onto an agarose-coated specimen holder using tissue glue (HistoAcryl; Braun) with the epicardial surface facing down. A high-precision vibrating microtome (7000 smz-2; Campden Instruments) was used to prepare 300-µm thin LMSs with a vibrating frequency of 80 Hz, amplitude of 2 mm, and advance speed of 0.03 mm/s. The orientation of the tissue block ensured that the ceramic blade cut in parallel to the fibers’ orientation of the LV to minimize the tissue damage (Fig. S1). Throughout the slicing process, the tissue was constantly submerged in ice-cold (4°C) oxygenated Tyrode’s solution containing 30 mM BDM. Different regions of the LV wall were studied as follow: subendocardial slices were obtained ≤600 µm from the endocardial surface, and subepicardial slices were obtained ≤600 µm from the epicardial surface. After preparation, LMSs were used immediately for culture experiments and randomly assigned to control or cryoinjury groups. Cryoinjury was performed using a 3-mm cylindrical rod made out stainless steel that was cooled down on dry ice (−78.5°C). The rod was placed carefully on the tissue for 2–3 s before the culture time and immediately removed, resulting in an injury.