Die dynamische Regulation von Protein-Protein Interaktionen (PPIs) ist wichtig für den Ablauf von biologischen Prozessen. Die circadiane Uhr, die einen ~24 Stunden Rhythmus generiert und eine Vielzahl von physiologischen Parametern steuert kann auch die Dynamik von PPIs regulieren. Um neue Erkenntnisse über regulatorische Mechanismen innerhalb des molekularen Oszillators zu gewinnen, habe ich zunächst alle möglichen PPIs zwischen 46 circadianen Komponenten mittels eines systematischen yeast-two-hybid (Y2H) Screens bestimmt. Dabei habe ich 109 bis dahin noch unbekannte PPIs identifiziert und einen repräsentativen Anteil mittels Co-Immunopräzipitationsexperimenten in humanen Zellen validiert. Unter den neuen PPIs habe ich bis dahin unbekannte Modulatoren der CLOCK/BMAL1 Transaktivierung identifiziert und dabei die Rolle der Proteinphosphatase 1 (PP1) als dynamischen Regulator der BMAL1 Stabilität funktionell charakterisiert. Das experimentelle PPI Netzwerk wurde mit bereits aus der Literatur bekannten PPIs und Interaktionspartnern ergänzt. Eine systematische RNAi Studie belegte außerdem die Relevanz der aus der Literatur stammenden Interaktoren für die ~24 Stunden Periodizität. Um eine Aussage über die Dynamik der PPIs im Netzwerk treffen zu können, wurden circadiane mRNA Expressionsdaten in das PPI Netzwerk integriert. Systematische Perturbationsstudien, in denen alle Komponenten des experimentellen Netzwerkes mittels RNAi herunterreguliert oder überexprimiert wurden, zeigten eine essentielle Bedeutung für die dynamischen PPIs innerhalb des circadianen Oszillators auf. Desweiteren wurden im circadianen PPI Netzwerk funktionelle Module identifiziert, welche dynamisch organsiert sind. Durch eine systemweite Analyse des humanen Proteoms wurden viele dynamische PPIs identifiziert, die biologische Prozesse wie z.B. Signaltransduktion und Zellzyklus miteinander verbinden. Rhythmische PPIs sind daher von Bedeutung für die zeitliche Organisation zellulärer Physiologie. / Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24 hrs) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes PPI dynamics on this timescale are largely unknown. To elucidate so far unknown regulatory mechanisms within the circadian clockwork, I have systematically mapped PPIs among 46 circadian components using high-throughput yeast-two-hybrid (Y2H) interaction experiments. I have identified 109 so far uncharacterized interactions and successfully validated a sub-fraction via co-immunoprecipitation experiments in human cells. Among the novel PPIs, I have identified modulators of CLOCK/BMAL1 function and further characterized the role of protein phosphatase 1 (PP1) in the dynamic regulation of BMAL1 abundance. Furthermore, to generate a more comprehensive circadian PPI network, the experimental network was enriched and extended with additional interactions and interaction partners from literature, some of which turned out to be essential for normal circadian dynamics. The integration of circadian mRNA expression profiles allowed us to determine the interaction dynamics within our network. Systematic genetic perturbation studies (RNAi and overexpression in oscillating human cells) revealed a crucial role of dynamic regulation (via rhythmic PPIs) for the molecular clockwork. Furthermore, dynamic modular organization as a pervasive circadian network feature likely contributes to time-of-day dependent control of many cellular processes. Global analysis of the proteome regarding circadian regulation of biological processes via rhythmic PPIs revealed time-of-day dependent organization of the human interactome. Circadian PPIs dynamically connect many important cellular processes like signal transduction and cell cycle, which contribute to temporal organization of cellular physiology.

A hipertensão arterial essencial é caracterizada pela elevação crônica da pressão arterial e representa o principal fator de risco para doenças cardiovasculares e renais. O rim participa do controle da pressão arterial e alterações intrínsecas no manuseio renal de sódio desempenham papel importante na patogênese da hipertensão essencial. Os túbulos proximais renais são responsáveis pela reabsorção da maior parte do sódio filtrado nos glomérulos e a maior parte da reabsorção de sódio neste segmento faz-se através da troca de Na+ por H+ em membrana apical, mediada pela isoforma 3 do trocador Na+/H+ (NHE3). Entretanto, os dados existentes referentes à modulação renal do NHE3 em modelos de hipertensão são ainda conflitantes. Este estudo teve como objetivo avaliar as possíveis alterações funcionais do trocador Na+/H+ NHE3 em túbulo proximal renal na linhagem SHR no estágio de préhipertensão (5 semanas) e de hipertensão (14 semanas) e investigar se estas alterações são acompanhadas de alterações na atividade e na expressão da proteína cinase A (PKA) e de proteínas fosfatase-1 (PP1). Por meio de microperfusão estacionária in vivo mediu-se a atividade do NHE3 em túbulo proximal e verificou-se que a reabsorção de bicarbonato foi reduzida em 62 ± 6 % (P < 0,001) na transição do J-SHR para o A-SHR enquanto foi aumentada em 113 ± 10 % (P < 0,001) na transição entre o J-WKY e o A-WKY. A atividade estimulada do NHE3 em J-SHR é decorrente da redistribuição do NHE3 do domínio intermicrovilar (IMV) para o domínio das microvilosidades (MMV) e do baixo nível de fosforilação da serina 552, sítio consenso para a PKA. Por outro lado, durante a fase de hipertensão, a atividade diminuída do NHE3 deve-se à sua redistribuição para o IMV e ao aumento da fosforilação na serina 552. Para testar a hipótese de que os níveis de fosforilação do NHE3 estariam aumentados em túbulo proximal de SHR adulto devido ao aumento da atividade da PKA e/ou à diminuição na atividade da PP1, foram avaliados tanto os níveis de fosforilação quanto a atividade do NHE3 em SHR jovens e adultos em resposta ao 6MB-cAMP (análogo ao cAMP que ativa especificamente a PKA). O JSHR apresentou um aumento tanto nos níveis de fosforilação da serina 552 (179 ± 14 %, P < 0,001) quanto nos de inibição da atividade (65 ± 10 %, P < 0,001) do NHE3 em relação ao J-SHR em resposta ao 6MB-cAMP. Já no A-SHR, a fosforilação da serina 552 aumentou moderadamente (36 ± 4 %, P < 0,01), assim como inibiu moderadamente (23 ± 9 %, P < 0,05) a atividade do NHE3 em resposta ao 6MBcAMP. Adicionalmente, verificou-se que não houve alteração da atividade da PKA entre os animais nem ao longo da idade e nem entre as linhagens. Por sua vez, o JSHR apresentou maior atividade da PP1 que o A-SHR (1640 ± 107 vs. 940 ± 119 pM/?g, P < 0,01). Além disso, houve uma diminuição na expressão da PP1? no ASHR (32 ± 8 %, P < 0,01) quando comparado ao J-SHR. Os dados sugerem que o NHE3 é diferencialmente regulado antes e após o desenvolvimento da hipertensão em SHR por mecanismos que envolvem modificações pós-transcricionais e distribuição subcelular. Além do mais, a regulação diferencial dos níveis de fosforilação do NHE3 tubular proximal antes e após o desenvolvimento da hipertensão em SHR é devida, provavelmente, a alterações na atividade e na expressão da PP1 / Essential hypertension is characterized by chronic elevation of blood pressure and represents the major risk factor for cardiovascular and renal diseases. The kidney participates in the blood pressure control and intrinsic changes in renal sodium handling play an important role in the pathogenesis of essential hypertension. The renal proximal tubule is responsible for reabsorption of the great majority of sodium that is filtered by the glomerulus and the principal apical membrane mechanism for sodium reabsorption in this nephron is Na+/H+ exchanger isoform 3 (NHE3)- mediated Na+/H+ exchange. However, conflicting data have been reported with regard to NHE3 modulation in experimental models of hypertension. This study aimed to evaluate the possible functional changes of the Na+/H+ exchanger NHE3 in the renal proximal tubule of SHR both at the pre-hypertensive (5 weeks) and at hypertensive (14 weeks) stages and to investigate whether these changes were accompanied by changes in the activity and/or expression of protein kinase A (PKA) and protein phosphatase 1 (PP1). Proximal tubule NHE3 activity was measured by means of stationary microperfusion. Bicarbonate reabsorption was found to be decreased by 62 ± 6 % (P < 0.001) in the transition from youth to adulthood in SHR (Y-SHR to A-SHR), whereas in the transition from Y-WKY to A-WKY it increased by 113 ± 10 % (P < 0.001). Stimulated NHE3 activity in Y-SHR was due to redistribution of NHE3 from intermicrovilar domain (IMV) to microvilar domain (MMV) and to a lower level of serine 552 phosphorylation, a consensus site for PKA. Conversely, during the hypertensive stage, decreased NHE3 activity was due to increased redistribution of NHE3 to the IMV domain and increased phosphorylation at serine 552. To test the hypothesis that the increased levels of NHE3 phosphorylation in the proximal tubule of adult SHR were due to increased PKA activity and/or decreased PP1 activity, it was evaluated both phosphorylation levels and activity of NHE3 in young and adult SHR in response to 6MB-cAMP (an cAMP analog that specifically activates PKA). Y-SHR showed an increase both in the phosphorylation levels at serine 552 (179 ± 14 %, P < 0.001) and in the inhibition of NHE3 transport activity (65 ± 10 %, P < 0.001) compared to Y-SHR in response to 6MB-cAMP. With respect to A-SHR, the phosphorylation of serine 552 was slightly increased (36 ± 4 %, P < 0.01) and NHE3 activity was mildly inhibited (23 ± 9 %, P < 0.05) in response to 6MB-cAMP. Additionally, PKA activity remained unchanged with both age and strain. Nevertheless, Y-SHR exhibited higher PP1 activity than A-SHR (1640 ± 107 vs. 940 ± 119 pM/?g, P < 0.01). Furthermore, PP1? expression was decreased in the renal cortex of A-SHR (32 ± 8 %, P < 0.01) compared to Y-SHR. Taken together, these data suggest that NHE3 is differentially regulated before and after development of hypertension in SHR by mechanisms involving post-translational modifications and subcellular distribution. Moreover, the differential regulation of proximal tubule NHE3 phosphorylation levels before and after development of hypertension in SHR is most likely due to changes on the activity and expression of PP1

Aims Protein Phosphatase Inhibitor 1 (I-1) functions as an amplifier of the β-adrenergic cascade in cardiomyocytes. Once activated via PKA, I-1 specifically blocks PP-1-mediated dephosphorylation of phospholamban and the ryanodine receptor-1. In heart failure I-1 activity as well as its expression is significantly reduced. It is still unclear whether this adaptation is protective or detrimental. This work aims at examining the impact of I-1 depletion on the course of pressure-induced heart failure, more precisely on acute and long-term mortality, on cardiac morphology and function and on expression levels of hypertrophy markers. Results may help evaluating the benefit of putative I-1 inhibiting substances in the therapy of heart failure. Methods and Results 25 I-1KO and 28 WT mice (C57Bl/6J, age- and sex-matched) underwent transverse aortic constriction (TAC). Cardiac function was assessed via transthoracic echocardiography prior to the intervention and weekly afterwards. Additionally, mice were exposed to β-adrenergic stimulation by injection of dobutamine once prior to TAC and two times afterwards, each controlled by echocardiography. For male mice acute survival was significantly increased in WT compared to I-1KO, whereas the mortality of surviving animals did not differ during the investigation period. For female mice no difference was seen in acute mortality after TAC, but during heart failure progression I-1KO revealed a significantly better survival. Prior to TAC contractility in I-1KO after application of dobutamine was significantly lower than in WT. This effect was mainly induced by female mice. Overall female mice of both WT and I-1KO showed smaller increases in heart rate (HR) and stroke volume (SV) when stimulated. In contrast, following TAC neither line- nor sex-dependent differences were found according to β-adrenergic stimulation. The comparison of hypertrophy markers in control groups revealed clearly decreased levels for I-1KO compared to WT. Conclusion In pressure-induced heart failure, I-1 knockout alters cardiac contractility and modulates mortality in a phase- and sex-dependent way. The depletion is detrimental for male mice in the acute phase of cardiac stress, whereas it is protective for female mice during heart failure progression. The increased mortality in the acute phase might result from the loss of I-1 as an amplifier of β-adrenergic signaling as this leads to a restriction of contractile adaptation. The increased survival in heart failure progression might be caused by a reduced transmission of pathologically increased sympathetic activity on the SR due to the depletion of I-1. Additionally, hypertrophy marker analyses point to differences in expression levels even under non-pathological conditions. / Ziel Der Proteinphosphatase-Inhibitor I-1 wirkt als ein Verstärker der β-adrenergen Kaskade in Kardiomyozyten. Nach PKA-abhängiger Phosphorylierung hemmt er spezifisch die Dephosphorylierung von PLB und RYR-2 durch die Proteinphosphatase-1. Im Rahmen einer Herzinsuffizienz sind sowohl Aktivität als auch Expression von I-1 deutlich reduziert. Hierbei ist unklar, ob dies eine protektive oder eine schädliche Adaption der β-adrenergen Kaskade darstellt. Diese Arbeit untersucht den Einfluss einer Depletion des I-1 (I-1KO) im Rahmen der druckinduzierten Herzinsuffizienz auf die akute bzw. auf die langfristige Mortalität, auf die kardiale Morphologie und Funktion sowie auf die Expression typischer Hypertrophiemarker. Hieraus sollen Erkenntnisse über den Nutzen der Verwendung putativ I-1 inhibierender Substanzen in der Behandlung der Herzinsuffizienz gewonnen werden. Methoden und Resultate 25 I-1KO- sowie 28 WT-Mäuse (C57Bl/6J, age and sex matched) erhielten eine Transverse Aortic Constriction (TAC). Die kardiale Funktion wurde einmalig vor der Intervention sowie danach wöchentlich mittels TTE untersucht. Zusätzlich wurden die Tiere einmalig vor TAC und zweimalig danach unter echokardiographischer Kontrolle mittels Dobutamin β-adrenerg stimuliert. Für die männlichen Tiere zeigte sich in den ersten Tagen nach TAC eine signifikant erhöhte Überlebensrate des WT gegenüber I-1KO. Die Mortalität der überlebenden männlichen Tiere unterschied sich hingegen nicht über den Versuchszeitraum. Für die weiblichen Tiere bestand kein Unterschied in der akuten Sterblichkeit nach TAC, während sich im Verlauf eine signifikant bessere Überlebensrate der weiblichen I-1KO gegenüber WT zeigte. Vor TAC wurde eine signifikant herabgesetzte Kontraktilität (FAS) des I-1KO unter Dobutamin festgestellt, der im Wesentlichen durch die weiblichen Tiere bewirkt wird. Insgesamt zeigten die weiblichen Tiere beider Linien unter β-adrenerger Stimulation eine geringere Zunahme von Herzfrequenz (HR) und Schlagvolumen (SV). Hingegen waren nach TAC keine linien- oder geschlechtsabhängigen Unterschiede unter Dobutamingabe feststellbar. Ein Vergleich der Hypertrophiemarker in der Kontrollgruppe zeigte für I-1KO ein deutlich vermindertes Niveau der Marker gegenüber WT. Ergebnis Der I-1-Knockout verändert die kardiale Kontraktilität und wirkt sowohl in phasen- als auch in geschlechtsabhängiger Weise auf die Mortalität infolge druckinduzierter Herzinsuffizienz. Er ist nachteilig für männliche Tiere in der akuten Phase kardialer Belastung, während er für weibliche Tiere im weiteren Verlauf protektive Wirkung entfaltet. Eine erhöhte Mortalität in der akuten Phase kann durch den Ausfall der Verstärkerfunktion des I-1 erklärt werden, da hiermit eine Einschränkung der akut notwendigen kontraktilen Adaptionsfähigkeit einhergeht. Ein Überlebensvorteil bei chronischer kardialer Belastung könnte darauf zurückzuführen sein, dass die pathologisch erhöhte sympathische Aktivierung der β-adrenergen Kaskade infolge der I-1-Depletion eine geringere Auswirkung auf die Zielstrukturen des aktivierten I-1 am Sarkoplasmatischen Retikulum hat. Darüber hinaus lassen die Analysen der Hypertrophiemarker eine veränderte Genexpression zwischen I-1KO und WT auch unter nicht-pathologischen Bedingungen vermuten.

A hipertensão arterial essencial é caracterizada pela elevação crônica da pressão arterial e representa o principal fator de risco para doenças cardiovasculares e renais. O rim participa do controle da pressão arterial e alterações intrínsecas no manuseio renal de sódio desempenham papel importante na patogênese da hipertensão essencial. Os túbulos proximais renais são responsáveis pela reabsorção da maior parte do sódio filtrado nos glomérulos e a maior parte da reabsorção de sódio neste segmento faz-se através da troca de Na+ por H+ em membrana apical, mediada pela isoforma 3 do trocador Na+/H+ (NHE3). Entretanto, os dados existentes referentes à modulação renal do NHE3 em modelos de hipertensão são ainda conflitantes. Este estudo teve como objetivo avaliar as possíveis alterações funcionais do trocador Na+/H+ NHE3 em túbulo proximal renal na linhagem SHR no estágio de préhipertensão (5 semanas) e de hipertensão (14 semanas) e investigar se estas alterações são acompanhadas de alterações na atividade e na expressão da proteína cinase A (PKA) e de proteínas fosfatase-1 (PP1). Por meio de microperfusão estacionária in vivo mediu-se a atividade do NHE3 em túbulo proximal e verificou-se que a reabsorção de bicarbonato foi reduzida em 62 ± 6 % (P < 0,001) na transição do J-SHR para o A-SHR enquanto foi aumentada em 113 ± 10 % (P < 0,001) na transição entre o J-WKY e o A-WKY. A atividade estimulada do NHE3 em J-SHR é decorrente da redistribuição do NHE3 do domínio intermicrovilar (IMV) para o domínio das microvilosidades (MMV) e do baixo nível de fosforilação da serina 552, sítio consenso para a PKA. Por outro lado, durante a fase de hipertensão, a atividade diminuída do NHE3 deve-se à sua redistribuição para o IMV e ao aumento da fosforilação na serina 552. Para testar a hipótese de que os níveis de fosforilação do NHE3 estariam aumentados em túbulo proximal de SHR adulto devido ao aumento da atividade da PKA e/ou à diminuição na atividade da PP1, foram avaliados tanto os níveis de fosforilação quanto a atividade do NHE3 em SHR jovens e adultos em resposta ao 6MB-cAMP (análogo ao cAMP que ativa especificamente a PKA). O JSHR apresentou um aumento tanto nos níveis de fosforilação da serina 552 (179 ± 14 %, P < 0,001) quanto nos de inibição da atividade (65 ± 10 %, P < 0,001) do NHE3 em relação ao J-SHR em resposta ao 6MB-cAMP. Já no A-SHR, a fosforilação da serina 552 aumentou moderadamente (36 ± 4 %, P < 0,01), assim como inibiu moderadamente (23 ± 9 %, P < 0,05) a atividade do NHE3 em resposta ao 6MBcAMP. Adicionalmente, verificou-se que não houve alteração da atividade da PKA entre os animais nem ao longo da idade e nem entre as linhagens. Por sua vez, o JSHR apresentou maior atividade da PP1 que o A-SHR (1640 ± 107 vs. 940 ± 119 pM/?g, P < 0,01). Além disso, houve uma diminuição na expressão da PP1? no ASHR (32 ± 8 %, P < 0,01) quando comparado ao J-SHR. Os dados sugerem que o NHE3 é diferencialmente regulado antes e após o desenvolvimento da hipertensão em SHR por mecanismos que envolvem modificações pós-transcricionais e distribuição subcelular. Além do mais, a regulação diferencial dos níveis de fosforilação do NHE3 tubular proximal antes e após o desenvolvimento da hipertensão em SHR é devida, provavelmente, a alterações na atividade e na expressão da PP1 / Essential hypertension is characterized by chronic elevation of blood pressure and represents the major risk factor for cardiovascular and renal diseases. The kidney participates in the blood pressure control and intrinsic changes in renal sodium handling play an important role in the pathogenesis of essential hypertension. The renal proximal tubule is responsible for reabsorption of the great majority of sodium that is filtered by the glomerulus and the principal apical membrane mechanism for sodium reabsorption in this nephron is Na+/H+ exchanger isoform 3 (NHE3)- mediated Na+/H+ exchange. However, conflicting data have been reported with regard to NHE3 modulation in experimental models of hypertension. This study aimed to evaluate the possible functional changes of the Na+/H+ exchanger NHE3 in the renal proximal tubule of SHR both at the pre-hypertensive (5 weeks) and at hypertensive (14 weeks) stages and to investigate whether these changes were accompanied by changes in the activity and/or expression of protein kinase A (PKA) and protein phosphatase 1 (PP1). Proximal tubule NHE3 activity was measured by means of stationary microperfusion. Bicarbonate reabsorption was found to be decreased by 62 ± 6 % (P < 0.001) in the transition from youth to adulthood in SHR (Y-SHR to A-SHR), whereas in the transition from Y-WKY to A-WKY it increased by 113 ± 10 % (P < 0.001). Stimulated NHE3 activity in Y-SHR was due to redistribution of NHE3 from intermicrovilar domain (IMV) to microvilar domain (MMV) and to a lower level of serine 552 phosphorylation, a consensus site for PKA. Conversely, during the hypertensive stage, decreased NHE3 activity was due to increased redistribution of NHE3 to the IMV domain and increased phosphorylation at serine 552. To test the hypothesis that the increased levels of NHE3 phosphorylation in the proximal tubule of adult SHR were due to increased PKA activity and/or decreased PP1 activity, it was evaluated both phosphorylation levels and activity of NHE3 in young and adult SHR in response to 6MB-cAMP (an cAMP analog that specifically activates PKA). Y-SHR showed an increase both in the phosphorylation levels at serine 552 (179 ± 14 %, P < 0.001) and in the inhibition of NHE3 transport activity (65 ± 10 %, P < 0.001) compared to Y-SHR in response to 6MB-cAMP. With respect to A-SHR, the phosphorylation of serine 552 was slightly increased (36 ± 4 %, P < 0.01) and NHE3 activity was mildly inhibited (23 ± 9 %, P < 0.05) in response to 6MB-cAMP. Additionally, PKA activity remained unchanged with both age and strain. Nevertheless, Y-SHR exhibited higher PP1 activity than A-SHR (1640 ± 107 vs. 940 ± 119 pM/?g, P < 0.01). Furthermore, PP1? expression was decreased in the renal cortex of A-SHR (32 ± 8 %, P < 0.01) compared to Y-SHR. Taken together, these data suggest that NHE3 is differentially regulated before and after development of hypertension in SHR by mechanisms involving post-translational modifications and subcellular distribution. Moreover, the differential regulation of proximal tubule NHE3 phosphorylation levels before and after development of hypertension in SHR is most likely due to changes on the activity and expression of PP1

Aims Protein Phosphatase Inhibitor 1 (I-1) functions as an amplifier of the β-adrenergic cascade in cardiomyocytes. Once activated via PKA, I-1 specifically blocks PP-1-mediated dephosphorylation of phospholamban and the ryanodine receptor-1. In heart failure I-1 activity as well as its expression is significantly reduced. It is still unclear whether this adaptation is protective or detrimental. This work aims at examining the impact of I-1 depletion on the course of pressure-induced heart failure, more precisely on acute and long-term mortality, on cardiac morphology and function and on expression levels of hypertrophy markers. Results may help evaluating the benefit of putative I-1 inhibiting substances in the therapy of heart failure. Methods and Results 25 I-1KO and 28 WT mice (C57Bl/6J, age- and sex-matched) underwent transverse aortic constriction (TAC). Cardiac function was assessed via transthoracic echocardiography prior to the intervention and weekly afterwards. Additionally, mice were exposed to β-adrenergic stimulation by injection of dobutamine once prior to TAC and two times afterwards, each controlled by echocardiography. For male mice acute survival was significantly increased in WT compared to I-1KO, whereas the mortality of surviving animals did not differ during the investigation period. For female mice no difference was seen in acute mortality after TAC, but during heart failure progression I-1KO revealed a significantly better survival. Prior to TAC contractility in I-1KO after application of dobutamine was significantly lower than in WT. This effect was mainly induced by female mice. Overall female mice of both WT and I-1KO showed smaller increases in heart rate (HR) and stroke volume (SV) when stimulated. In contrast, following TAC neither line- nor sex-dependent differences were found according to β-adrenergic stimulation. The comparison of hypertrophy markers in control groups revealed clearly decreased levels for I-1KO compared to WT. Conclusion In pressure-induced heart failure, I-1 knockout alters cardiac contractility and modulates mortality in a phase- and sex-dependent way. The depletion is detrimental for male mice in the acute phase of cardiac stress, whereas it is protective for female mice during heart failure progression. The increased mortality in the acute phase might result from the loss of I-1 as an amplifier of β-adrenergic signaling as this leads to a restriction of contractile adaptation. The increased survival in heart failure progression might be caused by a reduced transmission of pathologically increased sympathetic activity on the SR due to the depletion of I-1. Additionally, hypertrophy marker analyses point to differences in expression levels even under non-pathological conditions. / Ziel Der Proteinphosphatase-Inhibitor I-1 wirkt als ein Verstärker der β-adrenergen Kaskade in Kardiomyozyten. Nach PKA-abhängiger Phosphorylierung hemmt er spezifisch die Dephosphorylierung von PLB und RYR-2 durch die Proteinphosphatase-1. Im Rahmen einer Herzinsuffizienz sind sowohl Aktivität als auch Expression von I-1 deutlich reduziert. Hierbei ist unklar, ob dies eine protektive oder eine schädliche Adaption der β-adrenergen Kaskade darstellt. Diese Arbeit untersucht den Einfluss einer Depletion des I-1 (I-1KO) im Rahmen der druckinduzierten Herzinsuffizienz auf die akute bzw. auf die langfristige Mortalität, auf die kardiale Morphologie und Funktion sowie auf die Expression typischer Hypertrophiemarker. Hieraus sollen Erkenntnisse über den Nutzen der Verwendung putativ I-1 inhibierender Substanzen in der Behandlung der Herzinsuffizienz gewonnen werden. Methoden und Resultate 25 I-1KO- sowie 28 WT-Mäuse (C57Bl/6J, age and sex matched) erhielten eine Transverse Aortic Constriction (TAC). Die kardiale Funktion wurde einmalig vor der Intervention sowie danach wöchentlich mittels TTE untersucht. Zusätzlich wurden die Tiere einmalig vor TAC und zweimalig danach unter echokardiographischer Kontrolle mittels Dobutamin β-adrenerg stimuliert. Für die männlichen Tiere zeigte sich in den ersten Tagen nach TAC eine signifikant erhöhte Überlebensrate des WT gegenüber I-1KO. Die Mortalität der überlebenden männlichen Tiere unterschied sich hingegen nicht über den Versuchszeitraum. Für die weiblichen Tiere bestand kein Unterschied in der akuten Sterblichkeit nach TAC, während sich im Verlauf eine signifikant bessere Überlebensrate der weiblichen I-1KO gegenüber WT zeigte. Vor TAC wurde eine signifikant herabgesetzte Kontraktilität (FAS) des I-1KO unter Dobutamin festgestellt, der im Wesentlichen durch die weiblichen Tiere bewirkt wird. Insgesamt zeigten die weiblichen Tiere beider Linien unter β-adrenerger Stimulation eine geringere Zunahme von Herzfrequenz (HR) und Schlagvolumen (SV). Hingegen waren nach TAC keine linien- oder geschlechtsabhängigen Unterschiede unter Dobutamingabe feststellbar. Ein Vergleich der Hypertrophiemarker in der Kontrollgruppe zeigte für I-1KO ein deutlich vermindertes Niveau der Marker gegenüber WT. Ergebnis Der I-1-Knockout verändert die kardiale Kontraktilität und wirkt sowohl in phasen- als auch in geschlechtsabhängiger Weise auf die Mortalität infolge druckinduzierter Herzinsuffizienz. Er ist nachteilig für männliche Tiere in der akuten Phase kardialer Belastung, während er für weibliche Tiere im weiteren Verlauf protektive Wirkung entfaltet. Eine erhöhte Mortalität in der akuten Phase kann durch den Ausfall der Verstärkerfunktion des I-1 erklärt werden, da hiermit eine Einschränkung der akut notwendigen kontraktilen Adaptionsfähigkeit einhergeht. Ein Überlebensvorteil bei chronischer kardialer Belastung könnte darauf zurückzuführen sein, dass die pathologisch erhöhte sympathische Aktivierung der β-adrenergen Kaskade infolge der I-1-Depletion eine geringere Auswirkung auf die Zielstrukturen des aktivierten I-1 am Sarkoplasmatischen Retikulum hat. Darüber hinaus lassen die Analysen der Hypertrophiemarker eine veränderte Genexpression zwischen I-1KO und WT auch unter nicht-pathologischen Bedingungen vermuten.