Abstract-␤ 1 -and ␤ 2 -adrenergic receptors (␤ARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4ϾPDE2ϾPDE3. The ␤ 1 AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas ␤ 2 AR-mediated cAMP was under control of multiple PDE isoforms. ␤ 1 AR subtype-specific stimulation yielded Ϸ2-fold greater cAMP responses compared with selective ␤ 2 -subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (⌬FRET, 17.3Ϯ1.3% [␤ 1 AR] versus 8.8Ϯ0.4% [␤ 2 AR]). Treatment with pertussis toxin to inactivate G i did not affect cAMP production. Localized ␤ 1 AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local ␤ 2 AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, ␤ 1 ARs induce far-reaching cAMP signals, whereas ␤ 2 AR-induced cAMP remains locally confined. by the endogenous agonists noradrenaline and adrenaline represents the strongest mechanism to increase cardiac chronotropy and inotropy. 1 The mammalian heart contains 3 ␤AR subtypes: the ␤ 1 -, the ␤ 2 -, and the ␤ 3 AR. The ␤ 1 -and ␤ 2 AR subtypes dominate the cardiac response to adrenergic stimulation. Both are expressed in cardiomyocytes, couple primarily to G s , and mediate cAMP formation, whereas coupling of the ␤ 2 AR to G i has been described in several animal species and in failing human cardiomyocytes. [2][3][4] The second messenger cAMP then leads to activation of protein kinase A (PKA), which phosphorylates key regulators of the cardiac excitation/contraction machinery, including the L-type Ca 2ϩ channel, phospholamban, the ryanodine receptor, and troponin T and I. However, selective stimulation of these 2 receptor subtypes elicits different physiological responses. ␤ 1 AR stimulation, but not ␤ 2 AR stimulation, seems to induce cardiomyocyte hypertrophy. 5 Transgenic mice with cardiomyocyte-specific overexpression of the ␤ 1 AR develop progressive cardiac hypertrophy and heart failure, whereas ␤ 2 AR transgenic mice do not show such abnormalities. 6,7 Isolated cardio...

The extracellular-regulated kinases ERK1 and ERK2 (commonly referred to as ERK1/2) have a crucial role in cardiac hypertrophy. ERK1/2 is activated by mitogen-activated protein kinase kinase-1 (MEK1) and MEK2 (commonly referred to as MEK1/2)-dependent phosphorylation in the TEY motif of the activation loop, but how ERK1/2 is targeted toward specific substrates is not well understood. Here we show that autophosphorylation of ERK1/2 on Thr188 directs ERK1/2 to phosphorylate nuclear targets known to cause cardiac hypertrophy. Thr188 autophosphorylation requires the activation and assembly of the entire Raf-MEK-ERK kinase cascade, phosphorylation of the TEY motif, dimerization of ERK1/2 and binding to G protein betagamma subunits released from activated G(q). Thr188 phosphorylation of ERK1/2 was observed in isolated cardiomyocytes induced to undergo hypertrophic growth, in mice upon stimulation of G(q)-coupled receptors or after aortic banding and in failing human hearts. Experiments using transgenic mouse models carrying mutations at the Thr188 phosphorylation site of ERK2 suggested a causal relationship to cardiac hypertrophy. We propose that specific phosphorylation events on ERK1/2 integrate differing upstream signals (Raf1-MEK1/2 or G protein-coupled receptor-G(q)) to induce cardiac hypertrophy.

Abstract-Apoptosis of cardiomyocytes is increased in heart failure and has been implicated in disease progression. The activation of "proapoptotic" caspases represents a key step in cardiomyocyte apoptosis. In contrast, the role of "proinflammatory" caspases (caspases 1, 4, 5, 11, 12) is unclear. Here, we study the cardiac function of caspase-1. Gene array analysis in a murine heart failure model showed upregulation of myocardial caspase-1. In addition, we found increased expression of caspase-1 protein in murine and human heart failure. Mice with cardiomyocyte-specific overexpression of caspase-1 developed heart failure in the absence of detectable formation of interleukin (IL)-1␤ or IL-18 and inflammation. Transgenic caspase-1 induced primary cardiomyocyte apoptosis before structural and molecular signs of myocardial remodeling occurred. In contrast, deletion of endogenous caspase-1 was beneficial in the setting of myocardial infarction-induced heart failure. Furthermore, caspase-1-deficient mice were protected from ischemia/reperfusion-induced cardiomyocyte apoptosis. Studies in primary rat cardiomyocytes indicated that caspase-1 induces cardiomyocyte apoptosis primarily through activation of caspases-3 and -9. In contrast to previous findings, which imply a proinflammatory role of caspase-1, these data suggest a primary proapoptotic role for caspase-1 in cardiomyocytes. Our findings support a functional role for caspase-1-mediated myocardial apoptosis contributing to the progression of heart failure. (Circ Res. 2007;100:645-653.)

Hypertrophy represents the major physiological response of the heart to adapt to chronically enhanced workload, but is also crucial in the development of heart failure. Although we know of numerous inducers of cardiac hypertrophy, little is known about mechanisms that limit cardiac hypertrophy. Here, we describe the transcriptional repressor NAB1 as an endogenous regulator of cardiac growth. We identified NAB1 as being upregulated in both mouse and human heart failure. Nab1 is highly expressed in mammalian cardiac myocytes and it inhibited cardiomyocyte hypertrophy through repression of its targets, transcription factor Egr. Transgenic mice with cardiac-specific overexpression of Nab1 showed that Nab1 is a potent inhibitor of cardiac growth in response to pathological stimuli in vivo. Nab1 overexpression suppressed adrenergically induced and pressure overload-induced hypertrophy, whereas physiological growth during development and in response to exercise was not affected. These findings implicate the Nab1-Egr1 axis as a crucial regulator of pathological cardiac growth.

BackgroundIncreased activity of single ventricular L-type Ca2+-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary β-subunits as a possible explanation.Methods and ResultsBy molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC β-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac β-subunits: Unlike β1 or β3 isoforms, β2a and β2b induce a high-activity channel behavior typical of failing myocytes. In accordance, β2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac CaV1.2 also reveal increased single-channel activity and sarcolemmal β2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (“Adaptive Phase”), reveal the opposite phenotype, viz : reduced single-channel activity accompanied by lowered β2 expression. Additional evidence for the cause-effect relationship between β2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive CaV1.2 and inducible β2 cardiac overexpression. Here in non-failing hearts induction of β2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure.ConclusionsOur study presents evidence of the pathobiochemical relevance of β2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.