An arginine (R) to a glycine (G) mutation at position 145 in the highly reserved inhibitory domain of cardiac troponin I (cTnI) is associated with hypertrophic cardiomyopathy (HCM), an autosomal dominant disease characterized by left ventricular hypertrophy. An arginine (R) to tryptophan (W) mutation at the same position in cTnI is associated with restrictive cardiomyopathy (RCM), a disease characterized by diastolic dysfunction with normal left ventricular size and normal systolic function. In this study we addressed the functional consequences of the human cardiac troponin I (hcTnI) HCM R145G mutation and hcTnI RCM R145W mutation in transgenic mice. Simultaneous measurements of the ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca2+ activated force without changes in the maximal ATPase activity and an increase in the Ca2+ sensitivity by both ATPase activity and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state) showing that changes in Ca2+ sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared to Tg-WT fibers. The addition of 3mM BDM at pCa 9.0 showed that there was approximately 2~4 percent of force generating cross-bridges attached in Tg-R145G fibers compared to less than 1.0 percent in Tg-WT fibers, suggesting the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca2+] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared to Tg-WT. These results suggest that the phenotype of HCM is most likely caused by the compensatory mechanisms in the cardiovascular system which are activated by: 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge; 2) incomplete relaxation (diastolic dysfunction) caused by prolonged [Ca2+] and force transients; and 3) an inability of the cardiac TnI to completely inhibit activation at low levels of diastolic Ca2+ in Tg-R145G. Simultaneous measurements of the ATPase activity and force in transgenic skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) versus Tg-WT showed that there was a ~13 to ~16 percent increase in the maximal Ca2+ activated force and ATPase activity, respectively. The rate of dissociation of force generating cross-bridges (g) and energy cost (ATPase/force) was the same in all groups of fibers. These results suggest that the increase in force and ATPase activity is associated with an increase in the number of force generating cross-bridges attached at all activation levels. Additionally, there was a large increase in the Ca2+ sensitivity of force development and ATPase activity. In intact fibers, the mutation caused prolonged force and intracellular [Ca2+] transients, as expected due to the increased Ca2+ sensitivity (slower dissociation rate of Ca2+ from cTnC). The above cited results suggest that: 1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca2+ transients, especially at high heart rates; 2) there would be a decrease in ventricular filling (diastolic dysfunction); and 3) an increase in contractility during systole that would off-set the negative effect of a decrease in diastolic filling on ventricle stroke volume thus allowing the heart to maintain normal stroke volume despite the compromise in RCM (Tg-R145W) heart.

Cardiac tissue slices, a rarely used pseudo two-dimensional preparation, have gained increasing popularity for applications such as drug testing over the last ten years as they combine ease of handling with patho-physiologically relevant cell-type representation, distribution and inter-connection. The most well-established methods to measure electrophysiology in cardiac tissue are sharp electrodes and multi-electrode-arrays, techniques which are limited in spatial resolution or signal content. In this work, we have applied dual voltage Ca²⁺ optical mapping on cardiac slices, allowing us to record these two key parameters simultaneously at high spatio-temporal resolution, yielding better visualisation of conduction waves, spatial dispersion in action potential (AP) characteristics, and intracellular Ca²⁺ transient (CaT). The slice preparation method and the measurement protocols were refined to yield good reproducibility. Data analysis routines were developed to extract relevant parameters reliably. Despite being a promising candidate for drug testing, little is known about how slice and intact whole-heart AP properties are interrelated, and how to scale-up from observations in two dimensions (2D) to the three dimensional (3D) heart. In this thesis, we present a method to compare directly AP properties of intact whole-heart and tissue slices, and show the extent to which slices preserve AP characteristics. We have explored the suitability of tissue slices as an experimental model to study stretch induced changes in AP and CaT. During axial stretch, a dynamic profile of both AP and CaT was observed with an initial shortening of both AP and CaT duration, followed by a gradual recovery/prolongation. We have also used tissue slices to study spatial heterogeneity of AP and CaT properties in the rabbit left ventricular free wall. A transmural gradient can be captured in CaT and AP (with the longest APD and CaT durations being captured in the subendocardium). No large AP prolongation was found in the mid-myocardium. We conclude that the cardiac tissue slice preparation preserves some key functional parameters of the whole heart and is a promising model to study cardiac electrophysiology.

Familial hypertrophic cardiomyopathy (HCM) is a genetic disease largely caused by a mutation in myosin binding protein C (MYBPC3) and it affects about 1:500 population leading to arrhythmic sudden death, heart failure, and atrial fibrillation. MYBPC3 activates calcium-induced actin-myosin filament sliding within the cardiac sarcomere, creating the force necessary for heart contraction. The underlying molecular mechanisms causing HCM phenotype remain elusive, therefore, there is an urgent need for a reliable in vitro human HCM model to investigate the pathogenesis of HCM. This study utilized isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with MYBPC3 gene mutation (wildtype, heterozygous, homozygous) and further micropatterned them into fiber-like structures on polyacrylamide hydrogels of physiological and fibrotic-like stiffnesses. Cells were cultured for an extended culture time up to 60 days and their morphology/attachment, contractility, and calcium transient were extensively and carefully evaluated. It was found that MYBPC3 knockout cells maintained the highest contraction amplitude, but had increased contraction, and relaxation durations, decreased calcium transient amplitude, as well as time to peak and decay times over the culture period in comparison to the isogenic wildtype. Overall, this study demonstrates that hiPSC-CMs can be successfully patterned and cultured for an extended time on hydrogels forming end-to-end connections, which can be served as a simple yet effective in vitro human model for studying mechanical dysfunction of HCM.

No description available.

No description available.

Teas and infusions from C. spiralis leaf have largely been used by folk medicine as diuretic, hypotensor, cytotoxic, immunomodulator, antilithiasic, antidiarrheic, antispasmodic, antiurolitic, antimicrobian, antifungic, antioxidant, antileishmania activity, antiinflamatory, and antiedematogenic activity. In spite of these biological effects attributed to the extracts of C. spiralis, nothing so far could be found in the scientific literature dealing with its effects on the mammalian myocardium.The present study aimed to describe the inotropic effects produced by extracts from the C. spiralis leaf on isolated guinea pig atrium, as well as to contribute for a better understanding about its mechanism of action in that tissue. In isolated mouse cardiomyocytes, the effect produced by those extracts on the intracellular calcium transient and on the sarcolemal L-type calcium current were also measured. Experiments performed to evaluate the contractile effects were carried out on isolated atrium from guinea pig (Cavia porcellus). Firstly, our purpose was to determine the most potent fraction obtained from the C. spiralis leaf. This was done by comparing the hydroalchoolic crude extract with the following ones: aqueous, chloroform, and ethyl acetate. A phytochemical analysis was performed on the fraction exhibiting the greater potency. This evaluation followed the procedures proposed by Matos (1997). The content of sodium and potassium in the most potent fraction was determined by flame photometry. In the contractile experiments, the atrial force was measured isometrically. Biological signals were captured, amplified, and then stored in computer to be processed off line. Intracellular calcium transients were studied by confocal microscopy with laser scanning by using the fluorescent dye FLUO 4AM. Calcium inward currents were measured in mouse cardiomyocytes by using patch clamp technique in the whole cell configuration. Yield percentage of the aqueous fraction (AqF) was 69,40%. This fraction showed the most potent depressor effect on the myocardial contractility (EC50 = 305 ± 41,00 mg/L, Hill constant = 1,46 ± 0,19). The following metabolites were found in the AqF: tannins, saponins, and polifenols (flavonol, flavononol, flavone, xanthone, phenol, and flavonoid). The potassium and sodium contents in 1 g/L of AqF were 1,91 and 0,15 mM, respectively. This was not enough to change the myocardial inotropism, even in the highest concentration of AqF used in the experiments. The contraction and the relaxation time, as well as the time related to the excitation-contraction coupling (stimulus-response) were not modified by adding AqF to the organ bath. However, AqF reduced the Efficiency Index for the contraction and relaxation phases. The Neyler & Merrillees protocol was employed to evaluate the AqF effect on the calcium inward current in myocardial cells. Our results showed that AqF is able to completely abolish the Bowditch phenomenon, suggesting that it could be acting by reducing the sarcolemal calcium current. Supported by those experimental evidences, experiments were proposed to better understand the relationship between AqF and calcium mechanisms in cardiac cells. The following results were obtained with 1,5 g/L AqF: 1) AqF completely abolished the positive inotropic effect induced by isoproterenol (10-1 to 103 pM); 2) AqF shifted rightwardly the concentration-effect curve for CaCl2 (0.5 to 7.0 mM) and increased the EC50 from 1.12 ± 0.07 (Hill = 1.5) to 7.23 ± 0.47 mM (Hill = 7.4) (n = 3; p < 0.05); 3) AqF completely abolished the positive inotropic effect of (-) BAY K8644 (5 to 2000 nM); 4) AqF reduced the intracellular fluorescence from 4.66 ±1.17 to 3.74 ± 1.0 a.u. (n = 30 cells, 4 mice, p < 0.05); 5) AqF did not modify the decay rate of the fluorescent signal (892 ± 37 to 930 ± 30 ms, n = 30 cells, 4 mice, p > 0.05), indicating that it does not interfiere with the calcium removal from the sarcoplasm; 6) AqF reduced the calcium inward current through L-type calcium channels from 6,29 ± 0,34 to 4,9 ± 0,2 A/F (23% , n = 5 animals, p < 0,05). This study brought us unto the following conclusions: 1) AqF is the most potent fraction obtained from C. spirallis leaves; 2) AqF contains the following secondary metabolites: tannins, saponins, and poliphenols; 3) AqF reduces the contraction force of the guinea pig left atrium; 4) AqF acts on the myocardium contractility by reducing the calcium entry in myocardial cells during contraction. / Preparados de Costus spiralis têm sido usados pela medicina popular (diurético, hipotensor, citotóxico, imunomodulador, antilitiásico, antidiarréico, antiespasmódico, antiurolítico, antimicrobiano, antifúngico, antioxidante, antileishmânia, anti-inflamatório e antiedematogênico). Apesar da gama de ações a eles atribuídas, nada pôde ser encontrado na literatura científica com respeito ao possível efeito dos Este trabalho visou determinar os efeitos inotrópicos obtidos das folhas de C. spiralis, que apresentava maior potência, bem como contribuir para o mecanismo de ação desse preparado no miocárdio de mamíferos. Os experimentos sobre contração foram realizados em átrio esquerdo de cobaia (Cavia porcellus), enquanto que as medidas de transiente de cálcio intracelular e de corrente de membrana foram feitas em cardiomiócitos de camundongo. A investigação fitoquímica do preparado mais ativo foi conduzida segundo Matos (1997). Os teores de sódio e de potássio presentes na fração mais potente, foram determinados por fotometria de chama. A força de contração atrial foi captada isometricamente e, depois amplificada, foi armazenada em computador. O transiente de cálcio intracelular foi avaliado com microscopia confocal de varredura a laser. As correntes de cálcio sarcolemais foram medidas em cardiomiócitos submetidos à técnica do patch clamp ( whole cell ). A fração aquosa (FAq) foi a que apresentou maior rendimento (69,40%) e a que exerceu maior efeito inotrópico negativo (CE50 = 305 ± 41,00 mg/L, Hill = 1,46 ± 0,19). Na sua constituição foram detectadas as seguintes classes de metabólitos secundários: taninos e saponinas, com reação fortemente positiva, e os polifenóis, com reação positiva (flavonóis, flavononóis, flavonas, xantonas, fenóis e flavonóides). Em 1 g/L de FAq foram encontrados 1,91 mM de potássio e 0,15 mM de sódio. A adição de FAq ao Tyrode não modificou significativamente a concentração desses íons. Os tempos de contração e de relaxamento, bem como o tempo de acoplamento eletromecânico não foram alterados pela FAq. Contudo, ela reduziu os Índices de Eficiência da contração e do relaxamento. A FAq aboliu completamente o fenômeno de Bowditch induzido por alta frequência de estimulação, indicando que ela reduz a entrada desse íon nas células. Com base nessa evidência, foram realizados protocolos para aprofundar o conhecimento sobre a participação das correntes de cálcio no mecanismo cardiodepressor da FAq. Esta fração produziu os seguintes resultados: 1) aboliu completamente o efeito inotrópico positivo do isoproterenol (10-1 a 103 pM); 2) deslocou para a direita a curva concentração-efeito para o CaCl2 (0,5 a 7,0 mM), aumentando a CE50 de 1,12 ± 0,07 (Hill = 1,5) para 7,23 ± 0,47 mM (Hill = 7,4) (n = 3; p < 0,05); 3) aboliu completamente o efeito inotrópico positivo do (-) BAY K8644 (5 a 2000 nM); 4) reduziu em cerca de 20% o pico da fluorescência intracelular correspondente ao transiente de cálcio citoplasmático (controle: n = 30 células; teste: n = 27 células; 4 animais); 5) não modificou a velocidade de decaimento do sinal de fluorescência, o que significa que ela não interfere com o funcionamento da bomba de cálcio do retículo sarcoplasmático; 6) reduziu em 23% a densidade de corrente de cálcio tipo-L que variou de -6,29 ± 0,34 para -4,9 ± 0,2 A/F (n = 5 animais, p < 0,05). 1) a FAq foi a fração com maior potência inotrópica; 2) os principais metabólitos secundários presentes na FAq foram taninos, saponinas e polifenóis; 3) a FAq reduz a força de contração do átrio; 4) o mecanismo da ação cardiodepressora da FAq sobre a contratilidade miocárdica se deve à diminuição da disponibilização do cálcio durante a contração.

No description available.

Une accélération de la fréquence cardiaque (FC) au repos est observée chez les femmes enceintes. Au dernier trimestre, la FC accélère en moyenne de 15%, ce qui représente un facteur de risque dans le développement d’arythmies de novo ou dans l’exacerbation d’arythmies cardiaques préexistantes. Ceci est dangereux pour la mère ainsi que pour le fœtus. Cependant, les mécanismes responsables de ce changement cardiovasculaire restent peu connus. Notre laboratoire a récemment démontré que la grossesse était associée à une augmentation de la densité du courant pacemaker (If) et du courant calcique de type L (ICaL), ainsi qu’à des changements de l’homéostasie calcique dans les cellules de nœud sinusal (NS) de souris. Sachant que les concentrations plasmatiques en œstrogènes sont significativement augmentées pendant la grossesse et que ces hormones sexuelles féminines ont la capacité de modifier les propriétés électrophysiologiques du cœur, l’hypothèse de ce projet de recherche est que les œstrogènes jouent un rôle important dans l’augmentation de la FC associée à la grossesse et régulent les propriétés électrophysiologiques du NS. Les objectifs de ce projet de recherche sont de déterminer le rôle du 17β-œstradiol (E2) dans l’augmentation de la FC, d’examiner si ces effets sont régulés par les récepteurs aux œstrogènes alpha (ERα) et/ou bêta (ERβ) ainsi que d’évaluer les différents mécanismes de régulation de l’E2 sur l’électrophysiologie du NS. Des souris femelles adultes non-gestantes (2-4 mois) déficientes en ERα (ERKOα) ou en ERβ (ERKOβ) ont reçu un traitement chronique à l’E2 (30 μg deux fois par jour pendant quatre jours) simulant les concentrations plasmatiques en E2 retrouvées en fin de grossesse (23,3 ± 5,0 nM) chez la souris. L’analyse des électrocardiogrammes de surface montrent que la FC des souris ERKOβ (ERKOβ : 511 ± 15 bpm; ERKOβ +E2 : 580 ± 10 bpm, n = 10, p < 0,001) est significativement accélérée suivant le traitement à l’E2. Toutefois, la FC demeure inchangée chez les souris ERKOα (ERKOα : 520 ± 16 bpm; ERKOα +E2 : 530 ± 21 bpm, n = 7, p = 0,114). La méthode du patch-clamp en mode courant-imposé a permis de démontrer une accélération de l’automaticité des cellules du NS des souris ERKOβ suivant le traitement à l’E2, se traduisant par une augmentation de la fréquence des potentiels d’action spontanés (ERKOβ : 284 ± 24 bpm, n = 8; ERKOβ +E2 : 354 ± 23 bpm, n = 15, p = 0,0395) et par une pente de dépolarisation diastolique plus rapide (ERKOβ : 82 ± 12 mV/s, n = 8; ERKOβ +E2 : 140 ± 14 mV/s, n = 15, p < 0,003). En lien avec ces résultats, le patch-clamp en mode voltage-imposé a permis de démontrer que la densité de If est augmentée suivant un traitement à l’E2 (à -90 mV : ERKOβ : -6,6 ± 0,7 pA/pF, n = 12-15; ERKOβ +E2 : -11 ± 1 pA/pF, n = 9-11, p < 0,05). Cependant, If est similaire chez les souris ERKOα traitées ou non à l’E2. De plus, des cardiomyocytes humains dérivés de cellules souches pluripotentes induites de type nodal (N-hiPSC-CM) ont une accélération de la fréquence des potentiels d’action (CTL : 69 ± 5 bpm, n = 12; +E2 : 99 ± 6 bpm, n = 14, p < 0,001) ainsi qu’une augmentation de la densité de If (à -90 mV : CTL : -0,95 ± 0,14 pA/pF, n = 7-10; +E2 : -1,62 ± 0,17 pA/pF, n = 13-14, p < 0,05) suivant le traitement à l’E2. L’administration d’E2 ne modifie pas la fréquence des transitoires calciques des cellules de NS des souris ERKOα (139 ± 15, n = 13-14; +E2 : 142 ± 14, n = 15-16, p = ns) et ERKOβ (142 ± 11, n = 14-15; +E2 : 147 ± 13, n = 15-16, p = ns). En lien avec ces résultats, le courant ICaL des N-hiPSC-CM est inchangé suivant le traitement d’E2 (à 0 mV : CTL : -14,0 ± 1,3 pA/pF, n = 12-13; +E2 : -14,5 ± 1,4 pA/pF, n = 22, p = ns). En conclusion, l’accélération de l’automaticité cardiaque associée à la grossesse est, entre autres, expliquée par une augmentation de la densité de If, régulée par la voie de signalisation E2-ERα. Cependant, les changements de l’homéostasie calcique observés pendant la grossesse sont indépendants des niveaux élevés en œstrogènes. Les résultats obtenus sur les N-hiPSC-CM concordent avec ce qui est observé dans les cellules de NS de souris, ce qui démontre l’applicabilité humaine des résultats. Notre étude contribue à élucider l’influence de la grossesse et le rôle des hormones sexuelles féminines sur la fonction du NS et l’automaticité cardiaque. Ultimement, notre travail pourrait aider à développer une meilleure gestion des arythmies associées aux fluctuations hormonales féminines et/ou à la grossesse. / An increased heart rate (HR) is observed in pregnant women. In fact, in the last trimester, in average, the HR increases by 15%, which is a known risk factor to developing cardiac arrhythmias or exacerbating pre-existing arrhythmias. This can lead to major consequences for both the mother and fetus. However, the mechanisms underlying this increased HR remain largely unexplored. Our laboratory recently demonstrate that pregnancy is associated with an increased density of the pacemaker current (If) and the L-type calcium current (ICaL) as well as changes in calcium homeostasis of mouse sinoatrial node (SAN) cells. Knowing that estrogens are increased during pregnancy and that these sex hormones can modify cardiac electrophysiological properties, we hypothesized that estrogens play a key role in the pregnancy-induced increased HR and regulate the SAN electrophysiological properties. Our research project aims to determine the role of 17β-estradiol (E2) on the pregnancy-induced increased HR, to determine if these effects are regulated through estrogen receptor alpha (ERα) and/or beta (ERβ) and to study the E2 underlying mechanisms on SAN electrophysiology. Non-pregnant female mice (2-4 months) lacking ERα (ERKOα) or ERβ (ERKOβ) received a chronic E2 treatment (30 μg twice daily for four days) mimicking E2 concentrations found in late pregnancy (23.3 ± 5.0 nM). Surface electrocardiogram analysis showed a significant increased HR in ERKOβ mice (ERKOβ: 511 ± 15 bpm; ERKOβ +E2: 580 ± 10 bpm; n = 10; p<0.001) following E2 administration. However, the HR remains unchanged in ERKOα mice (ERKOα: 520 ± 16 bpm; ERKOα +E2: 530 ± 21 bpm, n = 7, p = 0.114). Following E2 treatment, current-clamp method demonstrates an increase SAN cells automaticity in ERKOβ mice, resulting in an increase in the spontaneous action potential frequency (ERKOβ : 284 ± 24 bpm, n = 8; ERKOβ +E2 : 354 ± 23 bpm, n = 15, p = 0.0395), associated with a steeper diastolic depolarization slope (ERKOβ : 82 ± 12 mV/s, n = 8; ERKOβ +E2 : 140 ± 14 mV/s, n = 15, p < 0.003), a major determinant of cardiac automaticity. In line with these results, voltage-clamp data showed an increased If density in SAN cells of ERKOβ mice treated with E2 (at -90 mV: ERKOβ: -6.6 ± 0.7 pA/pF, n = 12-15; ERKOβ +E2: -11.0 ± 1.3 pA/pF, n = 9-11, p < 0.05). Nevertheless, If density was similar in E2-treated ERKOα mice. E2-treated nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM) also showed an increased spontaneous action potential frequency (CTL : 69 ± 5 bpm, n = 12; +E2 : 99 ± 6 bpm, n = 14, p < 0.001) and If density (at -90 mV: CTL: -0.95 ± 0.14 pA/pF, n = 7-10; +E2: -1.62 ± 0.17 pA/pF, n = 13-14, p < 0.05). Following E2 administration, the rate of calcium transient was similar in SAN cells from ERKOα (139 ± 15, n = 13-14; +E2 : 142 ± 14, n = 15-16, p = ns) and ERKOβ (142 ± 11, n = 14-15; +E2 : 147 ± 13, n = 15-16, p = ns) mice. In line with these results, no modification was seen on ICaL density in E2-treated N-hiPSC-CM (at 0 mV: CTL: -14.0 ± 1.3 pA/pF, n = 12-13; +E2: -14.5 ± 1.4 pA/pF, n = 22, p = ns). In conclusion, the increased cardiac automaticity observed during pregnancy is, in part, explained by an increased If density. This mechanism is mediated by the E2-ERα pathway. In the other hand, calcium homeostasis changes detected during pregnancy appear to be mediated by an E2-independent mechanism. Finally, results obtained on N-hiPSC-CM are consistent with our observations on mouse SAN cells, demonstrating the human applicability of our results. This study provides novel insight on the effects of female sex hormones on the SAN functions. Ultimately, this information can lead to improved management of arrhythmias associated with female hormone fluctuations and/or pregnancy-induced arrhythmias.

Ahnak ist ein ubiquitäres Protein, das an einer Vielzahl biologischer Prozesse beteiligt ist. In der Herzmuskelzelle ist Ahnak überwiegend am Sarkolemma lokalisiert und bindet an Aktin und an die regulatorischen Beta2-Untereinheit des L-Typ-Kalzium-Kanals. Das Ziel dieser Arbeit war die Funktion von Ahnak im Herzen mit Hilfe eines Knock-out-Maus-Modells und in Bindungsstudien zu untersuchen. Morphologische Untersuchungen zeigten, dass das Längenwachstum adulter Kardiomyozyten bei Ahnakdefizienz signifikant reduziert war. Die Kontraktionseigenschaften adulter isolierter Ahnak-defizienter Kardio-myozyten (im Alter von 6 Monaten) waren ebenfalls verändert. Die Kontraktions- und Relaxaktionsgeschwindigkeiten waren erhöht. Eine Erhöhung des diastolischen Kalzium-Spiegels zeigten die Kardiomyozyten schon im Alter von 3 Monaten. Diese beobachteten phänotypischen Veränderungen lassen vermuten, dass die Aktivität des L-Typ-Kalzium-Kanals erhöht ist. In dieser Arbeit konnte das PXXP-Motiv, in der C-terminalen Ahnak-Domäne, als die hochaffine Beta2-Bindungsstelle (KD ~ 60 nM) identifiziert werden. Substitution von Prolin gegen Alanin verringerte zwar die Bindung zur Beta2-Untereinheit dramatisch (KD ~ 1 µM), hob sie aber nicht auf. In weiteren Bindungsstudien zeigte sich, dass die natürlich vorkommende Missensmutation I5236T die Bindung zur regulatorischen Beta2-Untereinheit verstärkte, dagegen verminderte die PKA-abhängige Phosphorylierung der beiden Proteinpartner die Bindung. Experimente am ganzen isoliert perfundierten Herzen zeigten, dass Ahnak-Knock-Out-Herzen geringer Beta-adrenerg stimulierbar waren. Ahnak scheint wie eine physiologische Bremse des kardialen Kalzium-Kanals zu wirken. / Ahnak is an ubiquitous protein with in unique structure, which has been implicated in cell type specific functions. In cardiomyocytes, ahnak is predominantly localized at the sarcolemma and is associated with actin and with the regulatory beta2 subunit of the L-type calcium-channel. The aim of this work was to unravel the function of ahnak in the heart, using a knock-out-mouse model and binding studies. Morphological studies showed a significant decrease in the cell-length of ahnak deficient cardiomyocytes. The contractile parameters of isolated adult ahnak deficient cardiomyocytes (in the age of 6 month) were altered. The development of tension and relaxation were increased. An increase of diastolic calcium was already observed at the age of 3 month. In general the observed phenotypic changes suggested an increased activity of the L-type calcium-channel. In this study, a PXXP-motif, which locates in ahnaks C-terminus, was identified as the high affinity beta2 subunit binding site (KD ~ 60 nM). Substitution of both proline residues by alanine reduced, but did not abolish the binding (KD ~ 1 µM). Further binding studies revealed that the natural occurring ahnak missense mutation I5236T increases the binding affinity to the regulatory beta2 subunit. By contrast PKA dependant phosphorylation of both protein partners decreases the interaction. In studies with isolated perfused working heart preparations, the ahnak deficient hearts were less beta-adrenergic stimulated than hearts from wild type. Taken together ahnak seems to be a physiological brake of the cardiac calcium-channel.