Protective Role Of PDE5 Inhibitor Sildenafil In Post-infarction Heart Failure Induced Mitochondrial Dysfunction And Its Mechanism

BackgroundCoronary artery diseases, particularly myocardial ischemia, are the most common cardiovascular disease worldwide. Although considerable improvements have made in therapeutics to drastically improve the survival rates, heart failure(HF) as the end stage of all cardiovascular disease remains a major cause of mortality and morbidity. During the past decades, HF treatment has made a great progress, however, the mortality of HF is still high with nowadays therapy and the development of HF can’t be restrained. In recent years cell energy metabolism is thought to play an important role in HF and is expected to be a novel target for the treatment of HF. Damaged mitochondrial ultrastructure and mitochondrial dysfunction were involved in HF induced by various causes. Mitochondrial dysfunction is the key reason responsible for myocardial energy hungry. How to reducethe damage of mitochondria, improve mitochondrial oxidative phosphorylation function is the key treatment for HF. Understanding the complex mechanisms underlying mitochondrial dysfunction is pertinent to the hunt for novel targets and therapeutic strategies to preserve mitochondrial integrity and optimize treatment and management for HF. The c GMP-specific phosphodiesterase-5(PDE5) inhibitors are approved clinically for treatment of erectile dysfunction and pulmonary arterial hypertension. However, a great amount of experimental and clinical studies have been implemented to explore the cardioprotection of PDE5 inhibitors. PDE5 inhibitors have been reported to attenuate myocardial ischemia injury and ameliorate the development of cardiac hypertrophy in pressure-overloaded hearts in mice with HF, reduce myocardial apoptosis and improve cardiac function. PDE5 inhibition enhances the accumulation of c GMP, and c GMP-dependent signaling has also been associated with mitochondria biogenesis and function in adipocytes. However, the effect of sildena?l on mitochondrial function, especially during post-infarction HF, remains unclear and needs to be further explored.Aims1. To identify whether PDE5 inhibitor, sildenafil could improve mitochondrial function in post-infarction HF and then attenuate left ventricular remodeling along with ameliorating cardiac dysfunction. 2. To explore the mechanism of sildenafil in the mitigation of mitochondrial ultrastructure and mitochondrial dysfunction in post-infarction HF. Evaluate Sirt3 and PGC-1α protein expression. 3. To determine the protective role of sildenafil in the hypoxia injury of cardiocytes in vitro and evaluate Sirt3/PGC-1α signal in the hypoxia injury protection; 4. To evaluate the role of sildenafil as a sirt3 activator for the therapeutic target against myocardial mitochondrial dysfunction in HF and other energy related disease.Methods1. Myocardial infarction was induced by the ligation of LAD. The cardiac function of each animal was assessed 3 days after MI to ensure similarities between groups and confirm the successful establishment of MI. Male C57BL/6 mice(8 weeks old) were randomized into two groups: MI+saline and MI+sildenafil. The mice received a 4-week treatment of either saline or sildena?l. Sham group was performed as control. Cardiac function was assessed at 7 and 28 days using M-mode echocardiography. 2. Cardiomyocytes were isolated from 1-day-old neonatal C57BL/6 mice, and were exposed to hypoxia for 24 h along with serum deprivation. 3. Sirt3 knockdown cardiomyocytes were eatablished via transfection with Sirt3 sh RNA plasmids. 4. At 28 days post infarction, animals were euthanized and their hearts were removed and then post-fixed in 4% paraformaldehyde to detect the fibrosis level by Masson’s trichrome staining and Sirius Red staining. The peri-infarct region of the heart was collected to measure the fibrosis gene markers MMP2 and MMP9. 5. At 28 days post infarction,the myocardium from the peri-infarct region was fixed with glutaraldehyde and cut into ultrathin sections to detect the mitochondrial ultrastructure using transmission electron microscopy. 6. Mitochondria isolated from the peri-infarct region of the heart were used to measure the oxygen consumption rate(OCR) and respiratory control ratio(RCR) using the Seahorse XF24 analyzer. 7. Mitochondria isolated from the peri-infarct zone was incubated with JC-1 or frozen section stained with JC-1 to determine the mitochondrial membrane potential(MMP) in vivo and in vitro cardiomyocytes incubated with JC-1 detected by flow cytometry. 8. The ATP content of the myocardium were measured using an ATP bioluminescent assay kit. 9. TUNEL staining, annexin V/PI flow cytometry and caspase-3 activity assay were performed to evaluate the apoptosis in vivo and in vitro. 10. Tissue specific non-radioactive PKG assay was used to measure PKG activity. 11. The m RNA expression of sirt3 was examined by RT-PCR analysis.12. The protein expression of PGC-1α, acetylated PGC-1α and sirt3 were examined by Western blot analysis.Results1. Compared to sham-operated mice, MI mice exhibited compromised cardiac function, as indicated by reduced percent LV shortening and ejection fractions. MI mice displayed enlarged LV cavities and severe LV remodeling. Cell apoptosis was increased in the border zone of MI mice, this indicated that cell apoptosis was an important factor contributing to HF. PDE5 inhibitor, sildenafil can attenuate and lessen the level of LV remodeling, cell apoptosis and improve cardiac function. 2. Most of mitochondria isolated from post-infarction HF displayed damaged mitochondrial ultrastructure, including disordered arrangement, cristae breakage, mitochondria swollen and even mitochondria vacuoles. Sildenafil alleviated the mitochondria ultrastructure disorder in HF hearts. A small number of swollen mitochondria was observed and no obvious vacuole damage was found in the group. Meantime, mitochondrial respiration function such as oxygen consumption rate and respiratory control ratio were impaired in the MI induced HF. Sildenafil was able to improve mitochondrial respiration function and mitochondrial membrane potential then increase ATP production. 3. Sirt3 expression was decreased in post-infarction HF, PGC-1α protein expression was not decreased as expected. However, PGC-α acetylation level was increased, this indicated PGC-α activity was decreased. Sildenafil treatment significantly increased Sirt3 expression and decreased PGC-1α acetylation in MI mice. 4. Cardiomyocytes exposed to hypoxia along with serum deprivation exhibited increased percentage of apoptotic cells, MMP collapse and decreased ATP production. Pretreatment with sildenafil partially alleviated the hypoxia injury. 5. Cardiomyocytes exposed to hypoxia along with serum deprivation exhibited attenuated Sirt3 expression and increased PGC-α acetylation level. Pretreatment with sildenafil partially revised the expression.6. Sirt3 knockdown increased percentage of apoptotic cells, MMP collapse and decreased ATP production, the protective role of sildenafil was partially attenuated by Sirt3 knockdown.Conclusions:1. PDE5 inhibitor, sildenafil can decrease cell apoptosis in post-infarction HF, lessen LV remodeling and improve cardiac function. 2. Sildenafil can prevent the mitochondrial ultrastructure damage, improve MMP collapse and mitochondrial function as well as ATP production. 3. The protective role of sildenafil in mitochondrial ultrastructure and mitochondrial function was mediated by upregulation of Sirt3 expression, downregulation of PGC-1α acetylation level and increased PGC-1α activity. 4. Sildenafil can protect cardiomyocytes from hypoxia injury. 5. Sirt3 knockdown make cardiomyocytes more susceptible to hypoxia injury. 6. Sirt3/ PGC-α signaling is involved in hypoxia induced cardiomyocytes injury and sildenafil reduces cardiomyocytes susceptibility to hypoxia injury, in part by upregulating Sirt3/ PGC-1α signaling.In conclusion, our research proves that the cardioprotective role of PDE5 inhibitor, sildenafil can be achieved by improving mitochondrial function, the protective role of sildenafil inmitochondria is mediated, in part, through a Sirt3 / PGC-1α signaling pathways, this finding provided potential target for mitochondrial energy metabolism in HF treatment and broaden the sildenafil use clinically to treat other energy related diseases.