All mice were of C57BL/6 background. They were maintained on a 12 h dark/light cycle (6 AM–6 PM) and housed in groups of ≤ 5 with unlimited access to water and chow. The experiments were performed with permission by the national Animal Experiment Board of Finland and by local authorities and conducted in accordance with the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Spry^fl/fl mice, that have been previously published [3], have Lox-P sites flanking open reading frame (ORF) in exon 3 of the mouse Spry1 gene The mice, backcrossed for > 7 generations, were crossed with Myh6-Mer-Cre-Mer mice (Jackson laboratories) in the same background and also backcrossed > 7 generations. Mer-Cre-Mer is constitutively expressed but is inactive until treatment with tamoxifen. At 7 weeks of age, the mice were injected with tamoxifen (40 mg/kg i.p., Cat 13258, Cayman Chemicals, Ann Arbor, MI) on 2 consecutive days. Analysis for genomic DNA from heart samples of Spry1^fl/fl × Myh6-Mer-Cre-Mer mice showed efficient Cre-lox recombination in the myocardium upon tamoxifen treatment (Online Fig. 1). At 8 weeks of age, cardiomyocyte Spry1 knockout (Spry1 cKO) and control mice were subjected to cardiac I/R injury.

Cardiac I/R injury was created by ligation of the left anterior descending artery (LAD) and releasing the slip knot after 30 min to allow for reperfusion of the myocardium as previously described in detail [12]. The mice were thereafter monitored for either 6 or 24 h and cardiac function and extent of cardiac injury (release of cTnI and infarct size or apoptosis) were analyzed. After the I/R procedure, mice were killed by cervical dislocation. Sham-operated mice were subjected to the same surgical procedure without ligation of the LAD. All operations were performed under isoflurane anesthesia (VetEquip vaporizer (VetEquip, Livermore, CA, USA), 2% isoflurane (Baxter, Deerfield, IL, USA), with 1 l/min flow of oxygen). Carprofen (Pfizer, New York, NY, USA) 5 mg/kg s.c. (injection volume 0.125–0.15 ml) was administered as pre-operation analgesia and buprenorphine (Orion Pharma, Espoo, Finland) 0.05 mg/kg s.c. (injection volume 0.1–0.3 ml) was administered as operation analgesia. Post-operation dehydration was prevented with s.c. injection of 5% glucose (injection volume 0.5–1.0 ml). All animals were monitored after the surgery and received a dose of buprenorphine (0.05 mg/kg) in the evening of operation day. Another dose of buprenorphine and a dose of carprofen (5 mg/kg) were administered the following morning.

Neonatal rat ventricular cardiomyocytes (CMs) were subjected to hypoxia on the fourth day after siRNA transfections. For normoxia cells, DMEM (11966-025, Gibco, Dublin, Ireland) supplemented with 10 mM glucose was used. For hypoxia cells, DMEM supplemented with 10 mM deoxyglucose and 1 mM sodium dithionite (Fluka, Buchs, Switzerland) was used. During hypoxia, cells were incubated in C-Chamber (BioSpherix, New York, NY, USA), with oxygen level controlled at 0.1% with ProOx C21 (BioPherix, New York, NY, USA). When needed 3 µM GSK-3β inhibitor SB216763 (1616, Tocris Bioscience, Bristol, United Kingdom) was added to CMs 20 min before hypoxia experiment.

Statistical analysis was performed with IBM SPSS Statistics software (IBM, Armonk, NY, USA). Normally distributed data were analyzed with one-way ANOVA followed by Tukey’s post hoc test. When two groups were compared, Student’s t test was used. If normality was not achieved, data were analyzed with Mann–Whitney test, or in case of three or more groups with Kruskal–Wallis followed by Dunn’s post hoc test. Differences were considered statistically significant at the level of P < 0.05. Data are shown as mean ± SD.

8-Week-old wild-type mice were subjected to experimental MI and protein samples from cardiac tissue were subjected to Western blot analysis. We found that MI induced an increase in levels of phosphorylated ERK and Spry1 protein in the infarcted areas of male myocardium compared to non-ischemic area (septum) that was more clear at 5 h after MI (Fig. 1a). Infarcted tissue of female mouse myocardium also showed induction in phosphorylated ERK and Spry1 expression analyzed 5 h after MI (Fig. 1a). On the other hand, myocardial infarction had no effect on expression of other cardiac Sprouty proteins, Spry2 or Spry4 (Fig. 1a).

We then investigated for Spry1 expression in resident cardiac cell types. In accordance with previous data [50], we found that Spry1 mRNA is expressed at eight-fold higher levels in fibroblast fraction compared to cardiomyocyte fraction in healthy hearts (Online Fig. 2). To assess if the increase in Spry1 protein expression occurs in cardiomyocytes in ischemic hearts, we subjected 8-week old male Spry1^fl/fl mice to I/R injury for 6 h and analyzed for localization of Spry1 expression by immunofluorescence microscopy. Analysis of cardiac sections showed that Spry1 is induced in ischemic areas in hearts of Spry1^fl/fl mice and colocalizes with cardiomyocyte marker α-actinin (Fig. 1b).

To investigate for the role of cardiomyocyte Spry1 in regulation of cardiac ischemic injury, we utilized tamoxifen-inducible Cre-lox technique to delete Spry1 specifically from the cardiomyocytes. Wild-type (WT), Spry1^fl/fl and Spry1 cKO mice were subjected to cardiac I/R injury with 30 min of ischemia followed by 24 h of reperfusion. Analysis of cardiac structure and function by echocardiography prior to cardiac ischemia showed no difference between WT, Spry1^fl/fl and Spry1 cKO mice (Online Table 1). Analysis for the extent of cardiac injury at 24 h after I/R showed that Spry1 knockdown in cardiomyocytes had no effect on area at risk, but significantly reduced infarct size compared to both WT and Spry1^fl/fl mice (Fig. 2a). Analysis of plasma samples showed that Spry1 cKO mice had significantly lower plasma cTnI levels at the 24 h after I/R injury compared to both WT and Spry1^fl/fl mice (Fig. 2b). Echocardiography analysis showed significantly better preserved systolic function in Spry1 cKO mice compared to WT mice, while the difference between Spry1 cKO and Spry1^fl/fl mice did not reach statistical significance (Table 1). No difference was observed in left ventricular end-diastolic diameter between the groups, but the stroke volume was greater in Spry1 cKO mice compared to Spry1^fl/fl or WT mice (Table 1). Spry1 cKO mice also showed a 20% increase in cardiac output compared to control mice (Table 1).

Spry1^fl/fl mice and Spry1 cKO mice were then subjected to cardiac I/R injury for 6 h. Analysis of plasma samples showed significantly lower plasma cTnI levels in Spry cKO mice compared to the Spry1^fl/fl mice (Fig. 2c). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of left ventricular (LV) sections showed a significant reduction in the number of apoptotic cells in ischemic areas of Spry1 cKO mouse hearts compared to the Spry1^fl/fl mouse hearts (Fig. 2c).

We next investigated if cardiomyocyte Spry1 deletion provides protection from I/R injury in female hearts. 8-Week-old female Spry1^fl/fl mice and Spry1 cKO mice were subjected to cardiac I/R injury for 24 h. Analysis for infarct size showed no difference in area at risk, but revealed a significant reduction in infarct size in female Spry1 cKO mice compared to female Spry1^fl/fl mice (Online Fig. 3a). Analysis of plasma samples of female mice showed a significant decrease in plasma cTnI levels in Spry cKO mice compared to the Spry1^fl/fl mice (Online Fig. 3b).

We then investigated if depletion of Spry1 in isolated cardiomyocytes offers protection from hypoxic injury. Spry1 RNAi in adult rat primary cardiomyocytes proved fruitless, since Spry1 mRNA expression was downregulated by 80–90% in non-treated healthy cardiomyocytes cultured for 2–4 days (Online Fig. 4). We then utilized RNAi in neonatal rat ventricular cardiomyocytes (CMs) which preserve their Spry1 expression in culture. Treatment of CMs with Spry1 siRNA led to 76% decrease in Spry1 mRNA and ~ 60% decrease in Spry1 protein levels (Fig. 3a). To investigate the effect of Spry1 RNAi on cardiomyocyte survival, Spry1 siRNA-treated CMs were subjected to simulated ischemia injury or cardiotoxic injury by doxorubicin treatment. Spry1-deficient cells showed markedly better survival [as assessed by release of adenylate kinase (AK) from ruptured cells] following 4 h of simulated ischemia (Fig. 3b). Spry1 knockdown, however, did not affect cardiomyocyte survival following treatment with 3 µM or 10 µM doxorubicin (Fig. 3c).

As Spry proteins have been shown to inhibit fibroblast growth factor induced Raf-ERK pathway activation [15], we assessed the effect of cardiomyocyte Spry1 knockdown on ERK activity. Western blot analysis for LV samples from healthy Spry1 cKO mice showed an increase in ERK phosphorylation in the myocardium compared to control Spry1^fl/fl mice (Fig. 4a, Online Fig. 5a). Analysis of ERK phosphorylation at 6 h after I/R injury showed that ERK phosphorylation remained elevated in ischemic myocardium of Spry1 cKO compared to ischemic areas of Spry1^fl/fl mouse hearts (Fig. 4a, Online Fig. 5a). In agreement with data from hearts in vivo, Spry1 knockdown in isolated cardiomyocytes increased ERK phosphorylation (Fig. 4b, Online Fig. 5b). Immunofluorescence analysis of cardiac sections showed that ERK is mainly phosphorylated in the vascular wall in hearts of sham-operated Spry1^fl/fl mice (Fig. 4c), whereas Spry1 cKO mice show ERK phosphorylation in cardiomyocytes (Fig. 4c). Immunofluorescence analysis after 6 h of I/R injury showed that ERK phosphorylation in cardiomyocytes is increased in both genotypes in ischemic areas, but Spry1 cKO cardiomyocytes show more intense staining (Fig. 4c). Spry1 thus negatively regulates ERK phosphorylation in cardiomyocytes at both baseline and following I/R injury.

Analysis of other central signaling pathways in cardiomyocytes showed that phosphorylation of p38 was increased in hearts of healthy Spry1 cKO mice compared to Spry^fl/fl mice (Fig. 4d, Online Fig. 5c). However, no difference was observed in p38 phosphorylation in ischemic tissues of Spry1^fl/fl and Spry1 cKO mice at 6 h after I/R injury (Fig. 4d, Online Fig. 5c). Analysis of other central cell survival pathways [phosphoinositide 3-kinase (PI3K)-Akt, Signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase] showed no difference between hearts of Spry^fl/fl and Spry1 cKO mice either prior to ischemia or after the ischemia. In accordance with in vivo findings, Spry1 knockdown in isolated neonatal cardiomyocytes showed increased p38 phosphorylation (Fig. 4e, Online Fig. 5d). Spry1 thus regulates p38 phosphorylation in non-injured cardiomyocytes.

Opening of the mitochondrial permeability transition pore (mPTP) at reperfusion is a critical determinant of I/R injury and inhibition of mPTP opening has been shown to reduce MI size and preserve cardiac function both in preclinical and clinical studies [5, 26]. To investigate if Spry1 regulates mPTP opening, we first investigated for Spry1 localization in injured cardiomyocytes. Analysis of LV sections from Spry1^fl/fl mice subjected to cardiac I/R injury for 6 h showed co-staining of Spry1 and mitochondrial marker heat shock protein 60 (HSP60) in cardiomyocytes (Fig. 5a). To determine if Spry1 regulates mitochondrial membrane potential (ΔΨm), Spry1 and control siRNA treated cardiomyocytes were subjected to 4 h of hypoxia and ΔΨm was measured with JC-1. We found that Spry1-deficient cardiomyocytes have significantly better preserved mitochondrial membrane potential compared to control cardiomyocytes (Fig. 5b). Similarly, analysis of mPTP opening by calcein cobalt assay showed better preserved mitochondrial membrane integrity in Spry1-deficient cardiomyocytes (Fig. 5b). Inhibition of GSK-3β has been shown to reduce mitochondrial membrane permeability transition and cell death, and ERK and p38 signaling pathways have both been shown to phosphorylate and inhibit GSK-3β [10, 25, 34, 49]. We then performed western blot analyses of pooled mitochondrial fractions from cardiomyocytes subjected to hypoxia and found that the levels of phosphorylated ERK and phosphorylated (inactive) GSK-3β were increased in mitochondria of Spry1-deficient cardiomyocytes compared to control siRNA transfected cardiomyocytes (Fig. 5c). To assess for possible role of GSK-3β in Spry1-mediated response, we transduced isolated cardiomyocytes with adenoviruses encoding for wild-type GSK-3β, GSK-3β harboring a Ser9Ala mutation and GSK-3β harboring Ser9Ala and Ser389Ala mutations. Phosphorylation of GSK-3β at Ser9 or at Ser389 has previously been shown to inactivate the kinase [48, 49]. We found that the forced activation of GSK-3β has no effect on hypoxia-induced cell death in control siRNA-treated cardiomyocytes (Fig. 5d). However, overexpression of GSK-3β (Ser9Ala) partially reverses the protective effect of Spry1 knockdown in hypoxic cardiomyocytes and overexpression of GSK-3β (Ser9Ala/Ser389Ala) completely abolishes the protective effect of Spry1 knockdown (Fig. 5d). Finally, we investigated if control and Spry1 siRNA-treated cardiomyocytes were sensitive to GSK-3β inhibition during hypoxia. We found that the treatment of control cardiomyocytes with SB216763 (3 μM) significantly reduces hypoxia-induced cardiomyocyte death (Fig. 5e). However, pharmacological inhibition of GSK-3β does not further protect Spry1-deficient cardiomyocytes from hypoxia-induced injury (Fig. 5e). These data thus indicate a crucial role for GSK-3β in regulating the protective response to Spry1 knockdown.