Here we describe a small family of proteins, termed MCIP1 and MCIP2 (for myocyte-enriched calcineurin interacting protein), that are expressed most abundantly in striated muscles and that form a physical complex with calcineurin A. MCIP1 is encoded by DSCR1, a gene located in the Down syndrome critical region. Expression of the MCIP family of proteins is up-regulated during muscle differentiation, and their forced overexpression inhibits calcineurin signaling to a muscle-specific target gene in a myocyte cell background. Binding of MCIP1 to calcineurin A requires sequence motifs that resemble calcineurin interacting domains found in NFAT proteins. The inhibitory action of MCIP1 involves a direct association with the catalytic domain of calcineurin, rather than interference with the function of downstream components of the calcineurin signaling pathway. The interaction between MCIP proteins and calcineurin may modulate calcineurin-dependent pathways that control hypertrophic growth and selective programs of gene expression in striated muscles.Calcineurin is a serine/threonine protein phosphatase that plays a pivotal role in developmental and homeostatic regulation of a wide variety of cell types (1, 2). The interaction of calcineurin with transcription factors of the NFAT 1 family following activation of the T cell receptor in leukocytes provides the best characterized example of how calcineurin regulates gene expression (3). Changes in intracellular calcium promote binding of Ca 2ϩ /calmodulin to the catalytic subunit of calcineurin (CnA), thereby displacing an autoinhibitory region and allowing access of protein substrates to the catalytic domain. Dephosphorylation of NFAT by activated calcineurin promotes its translocation from the cytoplasm to the nucleus, where NFAT binds DNA cooperatively with an AP1 heterodimer to activate transcription of genes encoding cytokines such as IL-2. This basic model of NFAT activation has been shown to transduce Ca 2ϩ signals via calcineurin in many cell types and to control transcription of diverse sets of target genes unique to each cellular environment (4). In each case, NFAT acts cooperatively with other transcription factors that include proteins of the AP1 (3), cMAF (5), GATA (6 -8), or MEF2 (9 -12) families. In addition to T cell activation, cellular responses controlled by calcineurin signaling include synaptic plasticity (11,13,14) and apoptosis (15,16).Recent studies of calcineurin signaling in striated myocytes of heart and skeletal muscle have expanded the scope of important physiological and pathological events controlled by this ubiquitously expressed protein. Forced expression of a constitutively active form of calcineurin in hearts of transgenic mice promotes cardiac hypertrophy that progresses to dilated cardiomyopathy, heart failure, and death, in a manner that recapitulates features of human disease (7). Moreover, hypertrophy and heart failure in these animals, and in certain other animal models of cardiomyopathy, are prevented by administration of the calcin...

Calcineurin, a calcium/calmodulin-regulated protein phosphatase, modulates gene expression in cardiac and skeletal muscles during development and in remodeling responses such as cardiac hypertrophy that are evoked by environmental stresses or disease. Recently, we identified two genes encoding proteins (MCIP1 and MCIP2) that are enriched in striated muscles and that interact with calcineurin to inhibit its enzymatic activity. In the present study, we show that expression of MCIP1 is regulated by calcineurin activity in hearts of mice with cardiac hypertrophy, as well as in cultured skeletal myotubes. In contrast, expression of MCIP2 in the heart is not altered by activated calcineurin but responds to thyroid hormone, which has no effect on MCIP1. A approximately 900-bp intragenic segment located between exons 3 and 4 of the MCIP1 gene functions as an alternative promoter that responds to calcineurin. This region includes a dense cluster of 15 consensus binding sites for NF-AT transcription factors. Because MCIP proteins can inhibit calcineurin, these results suggest that MCIP1 participates in a negative feedback circuit to diminish potentially deleterious effects of unrestrained calcineurin activity in cardiac and skeletal myocytes. Inhibitory effects of MCIP2 on calcineurin activity may be pertinent to gene switching events driven by thyroid hormone in striated muscles. The full text of this article is available at http://www. circresaha.org.

Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, ␣-myosin heavy chain promoter (␣-MHC). In unstressed mice, forced expression of hMCIP1 resulted in a 5-10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibited cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy that otherwise result from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibited hypertrophic responses to ␤-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure.

Abstract-Obesity-related diabetes mellitus leads to increased myocardial uptake of fatty acids (FAs), resulting in a form of cardiac dysfunction referred to as lipotoxic cardiomyopathy. We have shown previously that chronic activation of the FA-activated nuclear receptor, peroxisome proliferator-activated receptor ␣ (PPAR␣), is sufficient to drive the metabolic and functional abnormalities of the diabetic heart. Mice with cardiac-restricted overexpression of PPAR␣ (myosin heavy chain [MHC]-PPAR␣) exhibit myocyte lipid accumulation and cardiac dysfunction. We sought to define the role of the long-chain FA transporter CD36 in the pathophysiology of lipotoxic forms of cardiomyopathy. MHC-PPAR␣ mice were crossed with CD36-deficient mice (MHC-PPAR␣/CD36 Ϫ/Ϫ mice). The absence of CD36 prevented myocyte triacylglyceride accumulation and cardiac dysfunction in the MHC-PPAR␣ mice under basal conditions and following administration of high-fat diet. Surprisingly, the rescue of the MHC-PPAR␣ phenotype by CD36 deficiency was associated with increased glucose uptake and oxidation rather than changes in FA utilization. As predicted by the metabolic changes, the activation of PPAR␣ target genes involved in myocardial FA-oxidation pathways in the hearts of the MHC-PPAR␣ mice was unchanged in the CD36-deficient background. However, PPAR␣-mediated suppression of genes involved in glucose uptake and oxidation was reversed in the MHC-PPAR␣/ CD36 Ϫ/Ϫ mice. We conclude that CD36 is necessary for the development of lipotoxic cardiomyopathy in MHC-PPAR␣ mice and that novel therapeutic strategies aimed at reducing CD36-mediated FA uptake show promise for the prevention or treatment of cardiac dysfunction related to obesity and diabetes.

Summary Ca2+-activated BK channels modulate neuronal activities including spike frequency adaptation and synaptic transmission. Previous studies found that Ca2+ binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca2+ binding opens the gate over this distance remains unknown. By studying an Asp to Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca2+ binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca2+ sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca2+ binding to channel opening.