Advances in Imaging Technology for Cardiac Research55Establishing a model for intravital imaging of the beating murine heart56Altered RyR microdomains lead to more Ca2+ waves in non-coupled RyRs after myocardial infarction57Intravital microscopy (IVM), a new method for in vivo imaging of monocyte homing in a mouse hind limb arteriogenesis model
Authors: D Kavanagh, E Dries, C Baer, N Kalia, DJ Santiago, DM Johnson, P Claus, KR Sipido, W Zuschratter, M Riek-Burchardt, M Wagner, C Deffge, S Weinert, R Braun-Dullaeus, J Herold
Journal: Cardiovascular Research (2016)

# 55   Establishing a model for intravital imaging of the beating murine heart {#article-title-2}

Introduction: While stem cell (SC)-based therapy following heart injury holds promise, clinical success is compromised by poor SC recruitment to the heart following infusion. One solution is to identify strategies that may increase SC capture in cardiac microvessels. We have routinely used intravital microscopy (IVM), a method of live in vivo imaging of the microcirculation, to monitor cell trafficking. To do so in the heart would help facilitate research aimed at improving SC delivery. However, IVM of the beating mouse heart remains challenging due to difficulties with organ access and impractical degrees of tissue motion. The study presents a method that allows for cardiac IVM using a 3D-printed stabilizing device.

Methods: Anaesthetized mice were artificially ventilated using a small rodent ventilator which delivered medical oxygen. PEEP was applied by immersing the ventilator outlet in 4cm of ddH2O. A chest wall incision, expanded with retractors, facilitated the adhesive positioning of a specially designed tissue stabilizer onto the heart surface. This limited respiratory and cardiac contractile motion in a small area without compromising overall heart function. Imaging was performed through a small hole in the centre of the stabilizer. Preliminary studies were performed in the myocardial ischemia-reperfusion injury model. This injury was established by occlusion of the left anterior descending artery (LAD). The vessel was occluded for 45 minutes using a small piece of plastic tubing within a loop which was placed under the LAD. Identification of vascular dynamics (e.g. cell recruitment, monitoring of vascular no-reflow) took place at various points during reperfusion. Administration of FITC-BSA allowed for visualisation of the microvasculature. Endogenous neutrophils were labelled by intra-arterial administration of 6µg eFluor-660 labelled anti-Gr-1 antibody.

Results: Infusion of FITC-BSA permitted visualization of the coronary microcirculation. Vascular integrity was not disturbed and flow was visible as a result of application of the stabiliser. Endogenous (labelled) neutrophils were easily identified circulating in the cardiac microcirculation. Following cardiac IR, we identified significantly enhanced neutrophil recruitment as early as 60 minutes post-reperfusion, when compared to sham-operated control animals. Furthermore, using FITC-BSA visualization of the coronary microcirculation, we identified areas of vascular no-reflow following cardiac IR. Finally, systemically administered SCs (fluorescently labelled HSCs) could be identified trafficking through the cardiac microvasculature following cardiac IR injury.

Conclusion: We present a model for intravital imaging of the mouse beating heart in vivo. This method may be useful in monitoring cell dynamics and microvascular disturbances in the heart at the single cell level.

# 56   Altered RyR microdomains lead to more Ca2+ waves in non-coupled RyRs after myocardial infarction {#article-title-3}

Purpose: In ventricular myocytes of large mammals, a significant number of ryanodine receptors (RyRs) are not in couplons (i.e. non-coupled RyRs). Previous research from our lab has shown that these non-coupled receptors lack CaMKII and NOX2 microdomain modulation. Furthermore, after MI TTs are lost and hence the fraction of non-coupled RyRs is increased. We investigated whether this affected microdomain signaling and diastolic Ca2+ waves which are potentially arrhythmogenic.

Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=16), we studied myocytes from the area adjacent to the MI and compared these to myocytes from SHAM pigs (N=13) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator together with confocal line scan imaging. Myocytes were stimulated at a high frequency (2 Hz) in the presence of isoproterenol (10 nM; ISO). Ca2+ waves were recorded during a 15 s rest period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyRs using a specific algorithm.

Results: Conditioning myocytes at high frequency in the presence of ISO induced more Ca2+ waves in MI myocytes (0.21 ± 0.1 #waves/100µm/s; n=62) compared to SHAM myocytes (0.15 ± 0.1 #waves/100µm/s; n=59). No differences were found in wave foci, but a significant reduction in foci recruitment occurred after MI (57 % in MI vs. 70 % in SHAM). Moreover, in healthy myocytes more Ca2+ waves occurred in coupled compared to non-coupled in a 2:1 ratio. In MI myocytes, however, the subcellular site of wave occurrence was reversed to non-coupled sites resulting in a 1:2 ratio for coupled:non-coupled RyRs. Specific blockers directly (S107) or indirectly (AIP inhibiting CaMKII, mitoTEMPO scavenging mitochondrial ROS) targeting RyRs were investigated. In healthy myocytes, none of the blockers significantly affected wave frequency, neither at the global nor in the coupled versus non-coupled RyRs. However, in myocytes from MI animals, S107, mitoTEMPO and AIP all reduced the global increase in wave frequency. Subcellular analysis of different RyR subsets showed that in MI S107 (0.06 ± 0.1 #waves/100µm/s; n=27), AIP (0.06 ± 0.1 #waves/100µm/s; n=25) and mitoTEMPO (0.08 ± 0.1 #waves/100µm/s; n=22) reduced the wave frequency in non-coupled RyRs without significantly affecting coupled RyRs.

Conclusion: After MI, Ca2+ waves arise predominantly in non-coupled RyRs. The non-coupled RyRs after MI are modulated via local microdomains of CaMKII and mitochondrial ROS. These data highlight the arrhythmogenic nature of the non-coupled RyR clusters, which are at a higher density after MI, and suggest there may be a potential for selective pharmacological targeting.

# 57   Intravital microscopy (IVM), a new method for in vivo imaging of monocyte homing in a mouse hind limb arteriogenesis model {#article-title-4}

Therapeutic augmentation of collateral vessel growth (arteriogenesis) is of particular clinical interest. Monocytes are key players within this process. Quantification of collateralization in small animal models is, however, difficult and the commonly used techniques are post-mortem or histological assays. Live imaging of monocyte homing to sites of arteriogenesis in vivo has not been demonstrated until now. Therefore, an intravital microscopy (IVM) technique was established to visualize this process. The IVM is a non-invasive optical imaging technique offering high resolution, deep tissue penetration and video capturing. Nowadays only already located (homed) cells could be detected by post mortem examination of the tissue. Real time transmigration and interaction of cells had never been visualized under these circumstances.

By the use of this innovative and unique method we were able to examine the kinetics of monocyte homing in vivo and endogenous development of collateral arteries within the hind limb of C57BL6 mice after ligature of one femoral artery. The morphology of the collateral vessels in the adductor muscle was determined by 3D imaging and blood flow was visualized within the growing arteries. Our results were validated by histological workup and laser Doppler perfusion imaging (LDPI).

For the in vivo experiments, mice were anesthetized intraperitoneally and placed on a heating plate (37°C). Then, the leg was fixed between two adjustable stamps and care was taken to keep the region of interest moist. A cover glass was positioned on top of the stamps and adjusted before imaging was started. A Zeiss LSM 710 NLO with a 20x water immersion lens (NA 1.0) or a Leica SP 5 LSM microscope with a 10x dry lens (NA 0.4) were used in either 2 Photon or LSM mode, respectively. With both systems we were able to achieve deep tissue penetration up to 1 mm, thereby capturing multi-colour videos of living cells.

Histological workup of hind limb tissue sections of C57BL6 mice approved the increase of collateralization. The ratio of collateral arteries (ligated/unligated leg) was calculated (each group n=4). The intra individual ratio increased from 1.0±0.07 (prior to ligation) to 1.32±0.17 two weeks and 1.44±0.11 three weeks after ligation (P<0.05). Quantification of superficial skin perfusion by laser-Doppler perfusion imaging (LDPI, n=10) demonstrated a drop from 1.01±0.06 (ligated/unligated ratio) before ligature to 0.21±0.05 immediately after ligature as well. However, skin perfusion recuperated completely within one week to 0.94±0.17 and tended to be higher in the ligated than in the unligated hind limb after 3 weeks 1.02±0.11.

Our data demonstrate that IVM represents a potent tool for the visualization of collateral flow in small animal models and offers a new method for exploring intercellular interaction during the complex process of arteriogenesis.