Ruduwaan Salie

Effect of Sildenafil on Reperfusion Function, Infarct Size, and Cyclic Nucleotide Levels in the Isolated Rat Heart Model

We have previously shown that NO-donor induced elevation in myocardial cGMP levels is associated with improved reperfusion function of the isolated rat heart. The phosphodiesterase 5 (PDE 5) inhibitor, sildenafil could potentially increase myocardial cGMP levels and thus protect the heart against ischaemic/reperfusion injury.Methods: To test our hypothesis we treated the isolated working rat heart with vehicle, OR sildenafil (10, 20, 50, 100, 200 nM), OR sildenafil (50 nM) plus a sarcolemmal (HMR 1098) or a mitochondrial (5-Hydroxydecanoate (5-HD)) KATP channel blocker. Hearts were then subjected to 20 min global, or 35 min regional ischaemia at 37∘C before reperfusion function (aortic output, coronary flow and aortic pressure) and infarct size were documented. Pre-ischaemic, ischaemic and reperfusion myocardial cAMP and cGMP concentrations were determined.Results: Low concentrations of sildenafil (10, 20 and 50 nM) improved reperfusion aortic output (AO) recovery (61.4± 4.5%, 64.8 ± 5.2% and 62.3 ± 5.0% vs. 45.4 ± 3.8% for controls (p < 0.05)) and infarct size, while high concentrations (200 nM) worsened AO recovery (24.9 ± 4.9.0%, p < 0.05). Myocardial cGMP levels of ischaemic tissue were elevated (34.7 ± 2.4 vs. 27.3 ± 2.2 pmol/g ww) and cAMP levels were suppressed (0.59 ± 0.03 vs. 0.87 ± 0.06 nmol/g ww) in the sildenafil (50 nM) treated hearts. Co-perfusion with sildenafil plus HMR 1098 decreased AO recovery (21.7 ± 7.6% vs. 62.3 ± 5.0% for sildenafil alone, p < 0.05) and increased infarct size (29.7 ± 2.04% vs. 8.6 ± 2.39% for sildenafil alone, p < 0.05).Similarly, sildenafil plus 5-HD decreased reperfusion AO recovery (44.4 ± 6.0% vs. 62.3 ± 5.0% for sildenafil alone, p < 0.05) and increased infarct size (33.8 ± 1.62% vs. 8.6 ± 2.39% for sildenafil alone, p < 0.05).Conclusions: (1) Pretreatment with low concentrations of sildenafil (20–50 nM) improves, while higher concentrations (200 nM) worsen reperfusion function in this model. (2) Low concentrations of sildenafil (20–50 nM) decrease infarct size while the higher concentrations had no effect. (3) These protective properties of low concentrations of sildenafil may be related to its cGMP elevating and cAMP suppressing effects in the ischaemic heart. (4) Possible end-effectors for sildenafil in the ischaemic heart include the mitochondrial and sarcolemmal KATP channel.

The temporal relationship between p38 MAPK and HSP27 activation in ischaemic and pharmacological preconditioning

AbstractAn ischaemic preconditioning protocol and subsequent sustained ischaemia were characterized by activation and attenuation of p38 MAPK phosphorylation, respectively. However, the significance of events downstream of p38 MAPK needs investigation. Therefore the temporal relationship between phosphorylation of p38 MAPK and its downstream substrate HSP27 was studied during either an ischaemic or β–adrenergic preconditioning protocol and during sustained ischaemia.Isolated rat hearts were preconditioned (with or without a p38 MAPK inhibitor, SB203580) with 1 × 5 min or 3 × 5 min global ischaemia or 5 min β–adrenergic stimulation (10–7 M isoproterenol), followed by 25 min sustained ischaemia and 30 min reperfusion. Hearts were freeze–clamped at different time intervals and fractionated to determine p38 MAPK and HSP27 phosphorylation, via Western blotting.Significant phosphorylation of cytosolic p38 MAPK and membrane (myo–fibrillar) HSP27 occurred at the end of the first preconditioning episode. However, p38 MAPK phosphorylation disappeared during subsequent preconditioning episodes, while HSP27 phosphorylation was maintained for the duration of the protocol. Similar changes in p38 MAPK and HSP27 occurred with 5 min β–adrenergic preconditioning. After 25 min ischaemia, significant phosphorylation of cytosolic and membrane HSP27 was observed, while p38 MAPK phosphorylation was attenuated in ischaemic and β–adrenergic preconditioned compared to non–preconditioned hearts. SB203580–induced abolishment of p38 MAPK and HSP27 phosphorylation during the triggering phase of both preconditioning protocols reversed the changes in these parameters seen after sustained ischaemia.The results suggest that p38 MAPK activation triggers HSP27 phosphorylation during both the preconditioning protocols and during sustained ischaemia. Protection of preconditioned hearts during sustained ischaemia was characterized by phosphorylation of both cytosolic and myofibrillar HSP27.

ANG II type I receptor antagonism improved nitric oxide production and enhanced eNOS and PKB/Akt expression in hearts from a rat model of insulin resistance

Exogenous insulin therapy improves endothelial function in insulin resistant patients, indirectly indicating that nitric oxide synthase activity and NO production may be impaired. Insulin stimulates production of NO by activating a signaling pathway including insulin receptor substrate-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Angiotensin II type I (AT1) receptor-evoked oxidative stress is implicated in the inactivation of NO, impairing endothelium-dependent vasodilatation. Blocking the actions of Angiotensin II with an AT1 receptor antagonist (Losartan), has beneficial effects in patients with insulin resistance or type 2 diabetes mellitus. This study investigated whether elevated Angiotensin II influences myocardial insulin resistance, insulin signaling and NO production in a rat model of diet-induced obesity (DIO) by antagonizing the actions of the AT1 receptor with Losartan. Isolated, perfused hearts, Western blotting and flow-cytometric methods were utilized to determine myocardial function, expression and phosphorylation of key proteins and NO production, respectively. Results showed that hearts from DIO rats are insulin resistant (higher serine phosphorylation of IRS-1, lower insulin-stimulated phosphorylation of PKB/Akt and eNOS, lower NO production) and had poorer functional recovery and larger infarct development after ischaemia/reperfusion. Losartan improved the impaired functional recovery, and NO production and enhanced eNOS expression and phosphorylation and reduced infarct size in hearts from the DIO animals. Data obtained from Losartan treatment also revealed that Angiotensin II signaling modulates myocardial PKB/Akt expression. We conclude that Angiotensin II signaling exacerbates inhibition of NO production in insulin resistance and that this can be improved by AT1 antagonism.

The significance of the washout period in Preconditioning.

AIMS Exposure of the heart to 5 min global ischaemia (I) followed by 5 min reperfusion (R) (ischaemic preconditioning, IPC) or transient Beta 2-adrenergic receptor (B2-AR) stimulation with formoterol (B2PC), followed by 5 min washout before index ischaemia, elicits cardioprotection against subsequent sustained ischaemia. As the washout period during preconditioning is essential for subsequent cardioprotection, the aim of this study was to investigate the involvement of protein kinase A (PKA), reactive oxygen species (ROS), extracellular signal-regulated kinase (ERK), PKB/Akt, p38 MAPK and c-jun N-terminal kinase (JNK) during this period. METHODS Isolated perfused rat hearts were exposed to IPC (1x5min I / 5min R) or B2PC (1x5min Formoterol / 5min R) followed by 35 min regional ischaemia and reperfusion. Inhibitors for PKA (Rp-8CPT-cAMP)(16μM), ROS (NAC)(300μM), PKB (A-6730)(2.5μM), ERKp44/p42 (PD98,059)(10μM), p38MAPK (SB239063)(1μM) or JNK (SP600125)(10μM) were administered for 5 minutes before 5 minutes global ischaemia / 5 min reperfusion (IPC) or for 5 minutes before and during administration of formoterol (B2PC) prior to regional ischaemia, reperfusion and infarct size (IS) determination. Hearts exposed to B2PC or IPC were freeze-clamped during the washout period for Western blots analysis of PKB, ERKp44/p42, p38MAPK and JNK. RESULTS The PKA blocker abolished both B2PC and IPC, while NAC significantly increased IS of IPC but not of B2PC. Western blot analysis showed that ERKp44/p42 and PKB activation during washout after B2PC compared to IPC was significantly increased. IPC compared to B2PC showed significant p38MAPK and JNKp54/p46 activation. PKB and ERK inhibition or p38MAPK and JNK inhibition during the washout period of B2PC and IPC respectively, significantly increased IS. CONCLUSION PKA activation before regional ischaemia is a prerequisite for cardioprotection in both B2PC and IPC. However, ROS was crucial only in IPC. Kinase activation during the washout phase of IPC and B2PC, albeit different, affords the same cardioprotective response.