Christian Kuhn

The sarcomeric Z-disc: a nodal point in signalling and disease.

The perception of the Z-disc in striated muscle has undergone significant changes in the past decade. Traditionally, the Z-disc has been viewed as a passive constituent of the sarcomere, which is important only for the cross-linking of thin filaments and transmission of force generated by the myofilaments. The recent discovery of multiple novel molecular components, however, has shed light on an emerging role for the Z-disc in signal transduction in both cardiac and skeletal muscles. Strikingly, mutations in several Z-disc proteins have been shown to cause cardiomyopathies and/or muscular dystrophies. In addition, the elusive cardiac stretch receptor appears to localize to the Z-disc. Various signalling molecules have been shown to interact with Z-disc proteins, several of which shuttle between the Z-disc and other cellular compartments such as the nucleus, underlining the dynamic nature of Z-disc-dependent signalling. In this review, we provide a systematic view on the currently known Z-disc components and the functional significance of the Z-disc as an interface between biomechanical sensing and signalling in cardiac and skeletal muscle functions and diseases.

Mice lacking calsarcin-1 are sensitized to calcineurin signaling and show accelerated cardiomyopathy in response to pathological biomechanical stress.

Signaling by the calcium-dependent phosphatase calcineurin profoundly influences the growth and gene expression of cardiac and skeletal muscle. Calcineurin binds to calsarcins, a family of muscle-specific proteins of the sarcomeric Z-disc, a focal point in the pathogenesis of human cardiomyopathies. We show that calsarcin-1 negatively modulates the functions of calcineurin, such that calcineurin signaling was enhanced in striated muscles of mice that do not express calsarcin-1. As a consequence of inappropriate calcineurin activation, mice with a null mutation in calsarcin-1 showed an excess of slow skeletal muscle fibers. The absence of calsarcin-1 also activated a hypertrophic gene program, despite the absence of hypertrophy, and enhanced the cardiac growth response to pressure overload. In contrast, cardiac adaptation to other hypertrophic stimuli, such as chronic catecholamine stimulation or exercise, was not affected. These findings show important roles for calsarcins as modulators of calcineurin signaling and the transmission of a specific subset of stress signals leading to cardiac remodeling in vivo.

Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha

The degree of tumor load reduction as measured by cytogenetic response is an important prognostic factor for chronic myelogenous leukemia (CML) patients on therapy. We sought to determine whether BCR-ABL transcript levels can predict chromosomal response. Residual disease was evaluated in 120 CML patients in chronic phase (CP) treated with the selective tyrosine kinase inhibitor imatinib after resistance or intolerance to interferon α (IFN). Median time of therapy was 401 days (range 111–704). BCR-ABL and total ABL transcripts were measured in 486 peripheral blood (PB) specimens with a real time RT-PCR approach using fluorescent-labeled hybridization probes (LightCycler technology) and results were expressed as the ratio BCR-ABL/ABL. Cytogenetic response was determined in 3-monthly intervals: From 101 evaluable patients, 42 achieved a complete (CR, 0% Philadelphia chromosome (Ph)- positive metaphases), 18 a partial (PR, 1–34% Ph+), 13 a minor (MR, 35–94% Ph+), and 26 no response (NR, >94% Ph+). All PB samples were RT-PCR positive. The proportion of Ph+ metaphases and simultaneous BCR-ABL/ABL ratios correlated with ru2009=u20090.74, Pu2009<u20090.0001. In order to investigate whether early molecular analysis may predict cytogenetic response, quantitative RT-PCR data obtained after 1 and 2 months of therapy were compared with cytogenetic response at 6 months. BCR-ABL/ABL ratios after 1 month were not predictive, but results after 2 months correlated with the consecutive cytogenetic response (Pu2009=u20090.0008). The probability for a major cytogenetic response was significantly higher in patients with a BCR-ABL/ABL ratio <20% after 2 months of imatinib therapy. We conclude that: (1) quantitative determination of residual disease with real time RT-PCR is a reliable and sensitive method to monitor CML patients on imatinib therapy; (2) BCR-ABL/ABL ratios correlate well with cytogenetic response; (3) in IFN-pretreated patients all complete responders to imatinib have evidence of residual disease with the limited follow-up available; and (4) cytogenetic response at 6 months of therapy in CP patients is predictable with real time RT-PCR at 2 months.

Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy

Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptorxa0(FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated Txa0cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.

Gene Expression Pattern in Biomechanically Stretched Cardiomyocytes Evidence for a Stretch-Specific Gene Program

Biomechanical stress ie, attributable to pressure overload, leads to cardiac hypertrophy and may ultimately cause heart failure. Yet, it is still unclear how mechanical stress is sensed and transduced on the molecular level. To systematically elucidate the underlying signal transduction pathways, we analyzed the gene expression profile of stretched cardiomyocytes on a genome-wide scale in comparison with other inducers of hypertrophy such as pharmacological stimulation. Neonatal rat ventricular cardiomyocytes were either stretched biaxially or stimulated with phenylephrine (PE), both resulting in a similar degree of hypertrophy. Microarray analyses revealed 164 genes >2.0-fold up- and 21 genes <0.5-fold downregulated (P<0.01). Differential expression was confirmed by real-time polymerase chain reaction. Genes of the “fetal gene program” such as BNP were induced by both stretch (4.2×) and PE (2.9×). We also verified upregulation of known stretch-responsive genes, including HSP70 (20.9×) and c-myc (3.0×). Moreover, several genes were found to be preferentially induced by stretch, such as the cardioprotective cytokine GDF15 (24.8×) and heme oxygenase 1 (Hmox1, 10.8×; both confirmed on protein level). Neither PE nor endothelin-1 upregulated GDF15 and Hmox1, whereas angiotensin II significantly induced both genes. Conversely, the AT1 receptor blocker irbesartan markedly blunted stretch-mediated GDF15 and Hmox1 upregulation, suggesting that the angiotensin receptor tranduces the biomechanical induction of these genes. In conclusion, we report a comprehensive gene expression profile of cardiomyocytes subjected to biomechanical stress in comparison with pharmacologically induced hypertrophy. Our data imply that a stretch-specific gene program exists, which is mediated, at least in part, by angiotensin II–dependent signaling.

Detection and quantification of residual disease in chronic myelogenous leukemia.

The degree of tumor load reduction after therapy is an important prognostic factor for patients with CML. Conventional metaphase analysis has been considered to be the ‘gold standard’ for evaluating patient response to treatment but this technique normally requires bone marrow aspiration and is therefore invasive. The frequency of cytogenetic analyses can be considerably reduced if patients are also monitored by molecular methods, which can be performed on peripheral blood specimens. Of the various techniques available, most attention has been paid to RT-PCR for BCR-ABL mRNA since this is by far the most sensitive. Simple, non-quantitative RT-PCR analysis gives only limited information on patients after treatment. Quantitative RT-PCR assays have been developed to monitor the kinetics of residual BCR-ABL transcripts over time. Variables in the quantitative PCR assay may be controlled for by quantification of transcripts of a normal gene (eg ABL or glucose-6-phosphate dehydrogenase, G6PD) as an internal standard. After allogeneic stem cell transplantation, most patients become RT-PCR negative, often after a period of low level positivity that may persist for several months. Those patients destined to relapse are characterized by the reappearance and/or rising levels of BCR-ABL transcripts. In contrast, for patients treated with interferon-α (IFN) residual disease is rarely, if ever, eliminated. The actual level of minimal residual disease in complete cytogenetic responders to IFN correlates with the probability of relapse. New quantitative real time procedures promise to simplify the protocols that are currently in use, but standardization and the introduction of rigorous, internationally accepted controls are required to enable RT-PCR to become a robust and routine basis for therapeutic decisions.

Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation

The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle-specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2-deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.

Renal function and survival in 200 patients undergoing ECMO therapy

BACKGROUNDnExtracorporeal membrane oxygenation (ECMO) is increasingly used in the intensive care unit (ICU) setting to improve gas exchange in patients with acute respiratory distress syndrome as well as in patients pre- and post-heart and lung transplantation. In this clinical setting, acute kidney injury (AKI) is frequently observed. So far, it is unknown how AKI affects the survival of critically ill patients receiving ECMO support and whether veno-veno and veno-arterial ECMO have different effects on kidney function.nnnMETHODSnThis is a retrospective analysis of patients undergoing ECMO treatment in medical and surgical ICUs in a tertiary care centre. We evaluated all patients undergoing ECMO treatment at our centre between 1 January 2005 and 31 December 2010. Data from all 200 patients (83F/117M), median age 45 (17-83) years, were obtained by chart review. Follow-up data were obtained for up to 3 months.nnnRESULTSnThree-month survival of all patients was 31%. Of the 200 patients undergoing ECMO treatment, 60% (120/200) required renal replacement therapy (RRT) for AKI. While patients without RRT showed a 3-month survival of 53%, the survival of patients with AKI requiring RRT was 17% (P = 0.001). Longer duration of RRT was associated with a higher mortality.nnnCONCLUSIONSnAKI requiring RRT therapy in patients undergoing ECMO treatment increases mortality in ICU patients. Future studies have to clarify whether it is possible to identify patients who benefit from the combination of ECMO and RRT.

DYRK1A Is a Novel Negative Regulator of Cardiomyocyte Hypertrophy

Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors.

Calsarcin-1 Protects Against Angiotensin-II–Induced Cardiac Hypertrophy

Background— We have previously shown that deficiency for the z-disc protein calsarcin-1 (CS1) sensitizes the heart to calcineurin signaling and to stimuli of pathological hypertrophy. In the present study we asked whether overexpression of CS1 might exhibit antihypertrophic effects, and therefore we tested this hypothesis both in vitro and in vivo. Methods and Results— Adenoviral gene transfer of CS1 into neonatal cardiomyocytes inhibited hypertrophy as a result of Gq-agonist stimulation, including angiotensin-II (Ang-II), endothelin-1, and phenylephrine. Consistently, Adenoviral gene transfer of CS1 also led to the reduction of increased levels of atrial natriuretic factor (mRNA) and the calcineurin-sensitive gene MCIP1.4, suggesting that CS1 inhibits calcineurin-dependent signaling. Furthermore, we generated CS1-overexpressing transgenic mice (CS1Tg). Unchallenged CS1Tg mice did not exhibit a pathological phenotype as assessed by echocardiography and analysis of cardiac gene expression. Likewise, when subjected to long-term infusion of Ang-II, both CS1Tg and wild-type mice developed a similar degree of arterial hypertension. Yet, in contrast to wild-type mice, Ang-II–treated CS1Tg animals did not display cardiac hypertrophy. Despite the absence of hypertrophy, both fractional shortening and dP/dtmax were preserved in CS1Tg Ang-II–treated mice as assessed by echocardiography and cardiac catherization, respectively. Moreover, induction of the hypertrophic gene program (atrial natriuretic factor, brain natriuretic peptide) was markedly blunted, and expression of the calcineurin-dependent gene MCIP1.4 was significantly reduced in CS1Tg mice, again consistent with an inhibitory role of CS1 on calcineurin. Conclusions— The sarcomeric protein CS1 prevents Ang-II–induced cardiomyocyte hypertrophy at least in part via inhibition of calcineurin signaling. Thus, overexpression of CS1 might represent a novel approach to attenuate pathological cardiac hypertrophy.