Participação da via de sinalização da Proteína Cinase D (PKD1) sobre a regulação da reatividade vascular e a função do cardiomiócito ventricular

Abstract

The hormones and neurotransmitters of the renin angiotensin aldosterone system (RAAS) and the autonomic nervous system (ANS) can act on the vessels and heart, modulating several intracellular signaling pathways. Although the signaling pathways of the PKA and PKC enzymes are better studied and known, the signaling pathway of PKD has been little studied so far. PKD is an enzyme of the serine / threonine kinase family comprising three homologous PKD isoforms (PKD1 / PKCμ, PKD2 and PKD3 / PKCν) and belongs to the Ca2+ protein kinase dependent (CaMK) superfamily. The PKD1 isoform is increasingly recognized as a key regulator of a complex series of fundamental physiological processes, including: signal transduction, cell proliferation and differentiation, membrane transport, secretion, gene expression, use of substrate, and regulation of contractility myocardial. However, little is known about the role of PKD1 on vascular reactivity and cardiomyocyte regulation. Cardiomyocyte Study - Functional study performed on ventricular cardiomyocytes isolated from white New Zealand rabbits were transfected by adenoviruses called Kinase Activating Reporters (DKARs) that measure PKD activity in different locations of the cardiomyocytes (cytoplasm, nucleus and mitochondria). The DKARs in the nucleus showed higher values for the treatment made by aldosterone, whereas when we analyzed the cytoplasm and mitochondria, the performance of angiotensin II was observed in a more significant way. For the analysis of the calcium transient and contractility, ventricular cardimiocytes of C56BL6 mice were used, divided into groups: wild cardiac-specific WT and PKD1 KO (knockout). The main result was an increased response to angiotensin II in the cells of PKD WT animals when compared to cells without treatment (p = 0.0049) in the cytosol. The percentage of cardiomyocyte shortening was not different between groups, nor in the presence of drugs (angiotensin II, aldosterone and isoproterenol). Study of Vascular Function - Wistar rats (normotensive) and Wistar spontaneously hypertensive (SHR) were used. To assess whether the difference in systolic blood pressure (SBP) existed, measurement was performed using the tail plethysmography method (PAS Wistar 125.8 ± 1.2 mmHg and SHR = 204.8 ± 4.0 * mmHg, p < 0.01). Vascular reactivity was studied using isolated rings of the thoracic aorta to assess the contraction of the α-adrenergic stimulus (concentration curve response to Phenylephrine (10−10 to 3.5 x10−3 M) and the activation of AT receptors, using angiotensin II (10 −10 to 10−5 M), in the presence and absence: vascular endothelium; ICD 3.2 μM - a selective and specific inhibitor of PKD1; L-NAME 100 μM, a nonselective inhibitor of the enzyme nitric oxide synthase (NOS); the compound 1400W 1 μM, a potent and highly selective inhibitor of iNOS; N-ω-hydrochloride-propyl-Larginine 0.5 μM, a potent and selective inhibitor of nNOS. In the Wistar group, the contractile response to increasing doses of phenylephrine were not modified when PKD was inhibited with CID In the SHR group, we can identify a reduction in the contractile response in the presence of CID (Phenylephrine 10-3.5 M; CT = 116.6 ± 13.5 vs CID 60 ± 14.6%, p <0.05) .These data suggest the participation of the PKD pathway, in the opposite Useful induced by phenylephrine in the SHR group. Angiotensin II induced a contractile response, in a concentration-dependent manner, in the isolated aortic rings of all studied groups. In the Wistar group, the contractile response to angiotensin II was lower during incubation with DIC, while in the SHR group, PKD inhibition did not modify the contractile response to angiotensin II. In the Wistar group, iNOS inhibition increased the contractile response to angiotensin II when the PKD enzyme was inhibited, with the CID. In the SHR group, we can see that angiotensin IIdependent contractility was greater in the presence of the iNOS inhibitor, both in the presence and in the absence of PKD. These data suggest that, in the SHR group, NO production depends on the iNOS enzyme. In animals in the SHR group, inhibition of nNOS increased the contractile response to angiotensin II. Inhibition of the PKD pathway reduced the increase in the contractile response to angiotensin II during the inhibition of nNOS. These data suggest that, in the hypertensive group, the NO synthesis pathway, dependent on the nNOS enzyme, participates in the contractile response induced by angiotensin II. In conclusion, the set of results obtained in this study confirms our hypothesis of the participation of the PKD signaling pathway in the acute regulation of ventricular cardiomyocyte functions and in vascular reactivity. In ventricular cardiomyocytes, aldosterone acted directly on nuclear PKD together with HDACs, important regulators in cardiac remodeling. In the cytosol and mitochondria, angiotensin II acutely activated the PKD1 pathway. In relation to vascular reactivity, it was observed that in hypertensive animals, this role of modulating the signaling of the PKD pathway on vascular responses mediated by angiotensin II and phenylephrine, depends on the activation of different NOS isoforms, located in the vascular smooth muscle and endothelium. In the hypertensive group, modulation, via PKD, seems to depend more on the participation of nNOS, while in the normotensive group, on the pathways of eNOS and iNOS.