Activation of PKG and Akt Is Required for Cardioprotection by Ramelteon-Induced Preconditioning and Is Located Upstream of mKCa-Channels

Ramelteon is a Melatonin 1 (MT1)-and Melatonin 2 (MT2)-receptor agonist conferring cardioprotection by pharmacologic preconditioning. While activation of mitochondrial calcium-sensitive potassium (mK_Ca)-channels is involved in this protective mechanism, the specific upstream signaling pathway of Ramelteon-induced cardioprotection is unknown. In the present study, we (1) investigated whether Ramelteon-induced cardioprotection involves activation of protein kinase G (PKG) and/or protein kinase B (Akt) and (2) determined the precise sequence of PKG and Akt in the signal transduction pathway of Ramelteon-induced preconditioning. Hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. Before ischemia, hearts were perfused with Ramelteon (Ram) with or without the PKG or Akt inhibitor KT5823 and MK2206, respectively (KT5823 + Ram, KT5823, MK2206 + Ram, MK2206). To determine the precise signaling sequence, subsequent experiments were conducted with the guanylate cyclase activator BAY60-2770 and the mK_Ca-channel activator NS1619. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Ramelteon-induced infarct size reduction was completely blocked by KT5823 (p = 0.0012) and MK2206 (p = 0.0005). MK2206 with Ramelteon combined with BAY60-2770 reduced infarct size significantly (p = 0.0014) indicating that PKG activation takes place after Akt. Ramelteon and KT5823 (p = 0.0063) or MK2206 (p = 0.006) respectively combined with NS1619 also significantly reduced infarct size, indicating that PKG and Akt are located upstream of mK_Ca-channels. This study shows for the first time that Ramelteon-induced preconditioning (1) involves activation of PKG and Akt; (2) PKG is located downstream of Akt and (3) both enzymes are located upstream of mK_Ca-channels in the signal transduction pathway.