| dc.description.abstracteng | Heart failure is among the most common internal diseases in industrialized nations and associated with a high mortality rate. The sympathetic nervous system and β-adrenergic signaling play a crucial role in the pathophysiology of the insufficient heart. Initially, activation of the β-adrenergic pathway through the release of catecholamines stimulates the heart to use its cardiac reserve and maintain circulation and organ perfusion. Chronic β-adrenergic activation on the other hand leads into a pathophysiological vicious circle with a progressive decrease in cardiac function. In this context, cardiac remodeling and cardiac fibrosis as a cause of chronically elevated catecholamine levels are of significant importance. Protein phosphatase inhibitor-1 (I-1) is a downstream of cAMP embedded element within the β-adrenergic signaling pathway and functions as a β-adrenergic amplifier. At N-terminal Threonin 35 via PKA phosphorylated and thereby activated I-1 inhibits specifically protein phosphatase 1 and thus dephosphorylation of β-adrenergic target proteins. Earlier data of our group showed that heart-specific I-1-knock-out mice were partially protected from catecholamine-induced arrhythmias, cardiac hypertrophy and cardiac fibrosis induced by chronic β-adrenergic stress. In this context, extracellular matrix regulating cardiac fibroblasts and secretory highly active myofibroblasts represent the central effector cells in cardiac fibrosis. In this study, we analyzed the influence of β-adrenergic stimulation, in particular via overexpression of a constitutively active inhibitor-1 mutant (I-1c), on fibrosis associated proteins in cardiac fibroblasts (CF). Initially, it was unclear whether I-1 is endogenously expressed in CF. Our expression analysis data demonstrates an endogenous I-1 expression in CF of neonatal and adult mice (nMFB, aMFB), neonatal rats (nRFB) and adult humans (aHuFB) at mRNA level. While the highest I-1 mRNA levels were detected in CF of neonatal mice, the lowest mRNA levels were found in CF of adult humans (relative mRNA level of I-1 in nMFB approx. 20x higher compared to nRFB and approx. 200x higher compared to aHuFB). Interestingly, the ratio of endogenous I-1 expression differs species-specifically between CF and cardiomyocytes (higher I-1 expression in cardiomyocytes compared to CF in neonatal rats, higher I-1 expression in CF compared to cardiomyocytes in neonatal mice). In cardiomyocytes, conversion of I-1 in its inhibitory active form through PKA-depending phosphorylation at Threonin 35 is well known. In CF, however, it was unclear whether I-1 is also subject to β-adrenergic activation. Our data shows on protein level a concentration depending hyperphosphorylation and thereby activation of adenovirally overexpressed I-1 in CF of neonatal rats after β-adrenergic stimulation with the β-adrenergic agonist isoprenaline. Based on this, we could identify β2-adrenoceptors as the I-1 activating β-adrenoceptor subtype in CF. Contrary to our expectations, an influence of acute catecholamine stress through isoprenaline stimulation of CF on the transformation of fibroblasts to myofibroblasts could not be observed. In-vitro myofibroblast transformation by artificial cell culture conditions could be a possible reason in this matter. On the other hand, I-1 appears to favour fibroblast to myofibroblast transformation, a process essential for the development of cardiac fibrosis. Compared to control cells, I-1c overexpressing CF showed a significant increase in the expression of the myofibroblast marker α-smooth muscle actin (α-SMA). Furthermore, a tendency for an increased stress fiber formation in I-1c overexpressing CF could be observed in confocal microscopy. Also the expression of CTGF, a central mediator of fibrosis and remodeling, was slightly elevated in I-1c overexpressing CF. An influence of I-1 on the expression of collagen type I or its precursor protein procollagen α1 type in CF, however, could not be detected. In summary, I-1 was characterized as an element within the β-adrenergic pathway in cardiac fibroblasts and appears to exert profibrotic influences on cardiac fibroblasts through yet unknown mechanisms. In the context of heart failure, therapeutic modulation of I-1, in particular its inhibition, may have beneficial effects on cardiac remodeling and cardiac fibrosis. | de |