Norbert Frey

CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca(2+)-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy.

Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3β under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3β diminished hypertrophy in response to chronic β-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3β as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3β activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Decoding calcium signals involved in cardiac growth and function

Calcium is central in the regulation of cardiac contractility, growth and gene expression. Variations in the amplitude, frequency and compartmentalization of calcium signals are decoded by calcium/calmodulin-dependent enzymes, ion channels and transcription factors. Understanding the circuitry for calcium signaling creates opportunities for pharmacological modification of cardiac function.

Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy

Mitogen‐activated protein kinase (MAPK) pathways couple intrinsic and extrinsic signals to hypertrophic growth of cardiomyocytes. The MAPK kinase MEK5 activates the MAPK ERK5. To investigate the potential involvement of MEK5–ERK5 in cardiac hypertrophy, we expressed constitutively active and dominant‐negative forms of MEK5 in cardiomyocytes in vitro. MEK5 induced a form of hypertrophy in which cardiomyocytes acquired an elongated morphology and sarcomeres were assembled in a serial manner. The cytokine leukemia inhibitory factor (LIF), which stimulates MEK5 activity, evoked a similar response. Moreover, a dominant‐negative MEK5 mutant specifically blocked LIF‐induced elongation of cardiomyocytes and reduced expression of fetal cardiac genes without blocking other aspects of LIF‐induced hypertrophy. Consistent with the ability of MEK5 to induce serial assembly of sarcomeres in vitro, cardiac‐specific expression of activated MEK5 in transgenic mice resulted in eccentric cardiac hypertrophy that progressed to dilated cardiomyopathy and sudden death. These findings reveal a specific role for MEK5–ERK5 in the induction of eccentric cardiac hypertrophy and in transduction of cytokine signals that regulate serial sarcomere assembly.

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.

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.

Mechanisms of disease: hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most-common monogenically inherited form of heart disease, characterized by thickening of the left ventricular wall, contractile dysfunction, and potentially fatal arrhythmias. HCM is also the most-common cause of sudden cardiac death in individuals younger than 35 years of age. Much progress has been made in the elucidation of the genetic basis of HCM, resulting in the identification of more than 900 individual mutations in over 20 genes. Interestingly, most of these genes encode sarcomeric proteins, such as myosin-7 (also known as cardiac muscle β-myosin heavy chain; MYH7), cardiac myosin-binding protein C (MYBPC3), and cardiac muscle troponin T (TNNT2). However, the molecular events that ultimately lead to the clinical phenotype of HCM are still unclear. We discuss several potential pathways, which include altered calcium cycling and sarcomeric calcium sensitivity, increased fibrosis, disturbed biomechanical stress sensing, and impaired cardiac energy homeostasis. An improved understanding of the pathological mechanisms involved will result in greater specificity and success of therapies for patients with HCM.

Cardiac Z-disc Signaling Network

During the last 15 years, the perception of the cardiac z-disc has undergone substantial changes. Initially viewed as a structural component at the lateral boundaries of the sarcomere, the cardiac z-disc has increasingly become recognized as a nodal point in cardiomyocyte signal transduction and disease. This minireview thus focuses on novel components and recent developments in z-disc biology and their role in cardiac signaling and disease.

RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction.

AIMS Programmed necrosis (necroptosis) represents a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Like apoptosis, necroptosis is tightly regulated by distinct signalling pathways. A key regulatory role in programmed necrosis has been attributed to interactions of the receptor-interacting protein kinases, RIP1 and RIP3. However, the specific functional role of RIP3-dependent signalling and necroptosis in the heart is unknown. The aims of this study were thus to assess the significance of necroptosis and RIP3 in the context of myocardial ischaemia. METHODS AND RESULTS Immunoblots revealed strong expression of RIP3 in murine hearts, indicating potential functional significance of this protein in the myocardium. Consistent with a role in promoting necroptosis, adenoviral overexpression of RIP3 in neonatal rat cardiomyocytes and stimulation with TNF-α induced the formation of a complex of RIP1 and RIP3. Moreover, RIP3 overexpression was sufficient to induce necroptosis of cardiomyocytes. In vivo, cardiac expression of RIP3 was up-regulated upon myocardial infarction (MI). Conversely, mice deficient for RIP3 (RIP3(-/-)) showed a significantly better ejection fraction (45 ± 3.6 vs. 32 ± 4.4%, P < 0.05) and less hypertrophy in magnetic resonance imaging studies 30 days after experimental infarction due to left anterior descending coronary artery ligation. This was accompanied by a diminished inflammatory response of infarcted hearts and decreased generation of reactive oxygen species. CONCLUSION Here, we show that RIP3-dependent necroptosis modulates post-ischaemic adverse remodelling in a mouse model of MI. This novel signalling pathway may thus be an attractive target for future therapies that aim to limit the adverse consequences of myocardial ischaemia.