Supplemental Material

Methods

Mice

Conditional mice with floxed PKGI alleles (L2) were described earlier [9]. To generate cardiomyocyte specific PKGI knockout animals mice with floxed PKGI alleles (genotype: PKGIL2/L2) were crossed to the MLC2a-Cre transgenic mouse line (genotype: MLC2a-Cretg/+; PKGI+/L-). As shown earlier, the Cre-mediated recombination of the L2 allele produced the PKGI knockout (L-) specifically in atrial and ventricular cardiomyocytes but not in other tissues [9]. For experiments cardiomyocytes-specific PKGI knockout mice (CMG-KO; genotype: MLC2a-Cretg/+; PKGIL2/L-) were compared to control mice (CMG-CTR; MLC2a-Cretg/+; PKGI+/L2) from the same litters on a C57BL/6 genetic background. No changes in phenotype were observed between the different strains.

Open chest mouse model

We used an open-chest in situ mouse model as described earlier [1;7]. Mice were anaesthetized with pentobarbital sodium (70 mg/kg i.p.) and additional anaesthesia was administered as needed throughout the experiment. Animals were placed in supine position on a temperature-controlled heated table with rectal thermometer probe to maintain body temperature at 37°C. The trachea was surgically exposed, tracheal intubation was performed and correct tube placement was confirmed by direct visualization of the metal cannula. Animals were ventilated with room air supplemented with oxygen (peak inspiratory pressure of 10 mbar, positive end-expiratory pressure of 3 mbar). The ventilation frequency was set at 110 bpm and a tidal volume of 200–250 mL. After a left thoracotomy, exposure of the heart and dissection of the pericardium, a prominent branch of the left coronary artery was surrounded with a 7-0 nylon suture to form a snare. The coronary branch was occluded for 30 min respectively and reperfused for 2 h. To administer drugs either a butterfly needle was placed in the tail vein or in the left atrium. For determination of infarct size a double staining technique with Evans Blue and triphenyltetrazolium chloride (TTC) was used. Area at risk (AAR) was determined by retrograde injection of Evans Blue into the abdomen aorta while the LCA was re-occluded. All myocardial tissue was stained blue, except for AAR. The heart was excised, frozen, cut into 1 mm slides and slides were incubated with 1% TTC at 37°C for 15 min. The areas of infarct and risk zone were determined by planimetry of each slice and volumes calculated by multiplying each area by slice thickness and summing them for each heart. Infarct size is expressed as a percentage of the risk zone.

Experimental protocol

All hearts underwent 30 min coronary artery occlusion followed by 2 h of reperfusion. IPost was affected with six cycles of 10 sec reperfusion and 10 sec coronary artery occlusion following the index ischemia. BAY60 (10 μg/kg) was applied intraatrially as two separate boluses, 5 min before and 15 min after the onset of reperfusion. BAY58 (53.6 μg/kg) was given as an intravenous bolus 5 min before the onset of reperfusion followed by an infusion (1.25 μg/kg/min) for 65 min. MitoSNO and sildenafil (2 μg/kg) were administered as one bolus 5 min before the onset of reperfusion into the left atrium. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one (ODQ, 2 mg/kg) was given intraperitoneally 20 min before the onset of reperfusion. There was no mortality throughout the experiments. Hemodynamic effects of the drugs have been reported elsewhere [4;7;8]. In the present study we haven’t performed additional hemodynamic measurements since available catheter systems interfere with reliable infarct size measurements in mice.

Cardiac troponin I enzyme measurement

Cardiac troponin I (cTnI) is a standard marker for cardiac muscle damage and we measured cTnI levels in plasma at the end of reperfusion. Therefore, blood was obtained from the portals vein and centrifuged for 15 min at 4500 rpm. cTnI was determined by using a CTNI reagent kit and a Dimension Vista 1500, Integrated Analytics System (Siemens Healthcare Diagnostics, Deerfield IL).

Western Blot

Western Blot was performed as previously described [5]. ECL plex fluorescent secondary antibodies (dilution 1:2500, GE Healthcare) were applied to detect the complexes formed by the primary antibodies and antigens bound to a PVDF transfer membrane (Millipore). An EttanDIGE imager system (GE Healthcare) was used to visualize the immuno-complexes. The primary antibodies used were specific for PKGI common (dilution 1:200) [3], PKGIa (dilution 1:200) [2], GAPDH (Cell Signaling Technology) (dilution 1:1000), and a-actinin (Sigma-Aldrich) (dilution 1:1000).

Immunohistochemistry

Immunodetection was performed on paraffin embedded serial 8 μm sections of hearts obtained from CMG-CTR and CMG-KO mice according to a previously published protocol [6]. The primary antibody used for the IHC detected a common region of the PKGI protein (dilution 1:50) [3].

Materials

BAY60 and BAY58 were kindly provided by Bayer HealthCare (Wuppertal, Germany) while sildenafil was from Sigma-Aldrich Chemical Co. Drugs were dissolved in DMSO before being diluted in 0.9% NaCl. Final concentration of DMSO was kept below 0.01%. MitoSNO was synthesized as described previously [8]. Solutions were stored in absolute ethanol at −80°C and diluted in 0.9% NaCl before administration.

References

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