R. Sanders Williams

Cytochrome c Deficiency Causes Embryonic Lethality and Attenuates Stress-Induced Apoptosis

Cytochrome c released from mitochondria has been proposed to be an essential component of an apoptotic pathway responsive to DNA damage and other forms of cell stress. Murine embryos devoid of cytochrome c die in utero by midgestation, but cell lines established from early cytochrome c null embryos are viable under conditions that compensate for defective oxidative phosphorylation. As compared to cell lines established from wild-type embryos, cells lacking cytochrome c show reduced caspase-3 activation and are resistant to the proapoptotic effects of UV irradiation, serum withdrawal, or staurosporine. In contrast, cells lacking cytochrome c demonstrate increased sensitivity to cell death signals triggered by TNFalpha. These results define the role of cytochrome c in different apoptotic signaling cascades.

MEF2 responds to multiple calcium‐regulated signals in the control of skeletal muscle fiber type

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin‐dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium‐regulated signaling inputs, specifically calmodulin‐dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber‐specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up‐regulates a MEF2‐dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained.

Activation of MEF2 by muscle activity is mediated through a calcineurin‐dependent pathway

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. Here we observed that the functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in trans genic mice of a MEF2‐dependent reporter gene (desMEF2‐lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein, myocyte‐enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction.

Calcineurin signaling and muscle remodeling.

Increasingly, research into the mechanisms for muscle remodeling has revealed both new components of individual signaling pathways and the interactive participation of parallel pathways that converge on common downstream effector molecules and on common target genes. With respect to new components of calcineurin signaling pathways in cardiac and skeletal muscles, several recent findings begin to shed light on previously irreconcilable sets of data. For example, the ability of calcineurin to control the functional activity of MEF2 transcription factors, in addition to its effects on NFAT, explains how expression of certain genes can be modulated by calcineurin-dependent signaling in the absence of NFAT binding sites within promoter/enhancer regions (1xCalvo, S., Venepally, P., Cheng, J., and Buonanno, A. Mol. Cell Biol. 1999; 19: 515–525See all References, 20xWu, H., Naya, F.J., McKinsey, T.A., Mercer, B., Shelton, J.M., Chin, E.R., Simard, A.R., Michel, R.N., Bassel-Duby, R., Olson, E.N., and Williams, R.S. EMBO J. 2000; 19: 1963–1973Crossref | PubMedSee all References). The link between calcineurin and MEF2 also may account, at least in part, for recent observations that support a role for calcineurin in promoting myogenic differentiation during development (Friday et al. 2000xFriday, B.B., Horsley, V., and Pavlath, G.K. J. Cell Biol. 2000; 149: 657–665Crossref | Scopus (175)See all ReferencesFriday et al. 2000). Proteins have also been discovered recently that bind calcineurin and inhibit its protein phosphatase activity, thereby providing another means by which calcineurin signaling can be controlled. Two of these—Cabin/Cain and AKAP79—are expressed ubiquitously, while others—MCIP proteins—are expressed selectively in striated muscles. Such inhibitory mechanisms that alter calcineurin signaling may influence responses to calcineurin antagonist drugs in a manner that varies among different tissues.An important question is whether the hypertrophic and gene regulatory effects of calcineurin in striated myocytes are mediated directly by downstream transcription factors like NFAT and MEF2, or whether other substrates are responsible for the effects of calcineurin. Notably, calcineurin is associated with the 1,4,5-trisphosphate (IP3) and ryanodine receptors and regulates expression of the IP3 receptor and plasma membrane Ca2+ pump, which would be expected to have significant effects on Ca2+ handling, contractility, and probably hypertrophic growth (Crabtree 1999xCrabtree, G.R. Cell. 1999; 96: 611–614Abstract | Full Text | Full Text PDFSee all ReferencesCrabtree 1999). It may also be instructive to consider the functions of calcineurin in yeast, where it regulates Ca2+ homeostasis and stress responsiveness by controlling target genes involved in ion fluxes (Stathopoulos and Cyert 1997xStathopoulos, A.M. and Cyert, M.S. Genes Dev. 1997; 11: 3432–3444Crossref | PubMedSee all ReferencesStathopoulos and Cyert 1997). Whether calcineurin acts similarly in muscle cells remains to be determined.In summary, rapid progress has been made in delineating the signaling pathways that influence muscle remodeling responses to environmental or pathological stresses, and multiple layers of regulatory complexity to the pathways for remodeling myocytes in response to intrinsic and extrinsic signaling have been identified (Figure 1Figure 1). Subtle differences in the relative timing or intensity of individual primary signals, in specific features of the metabolic milieu or initial state of the cell, or in genetic background may lead to differences in the degree to which any single pathway is activated to produce similar remodeling responses (e.g., cardiac hypertrophy). This discovery process will continue, but the next phase of work in this field is likely to turn more to the task of unraveling molecular details of the complex interactions among diverse sets of signals, transducers, effectors, primary responses, and secondary responses that ultimately produce different remodeling outcomes in cardiac and skeletal muscles.‡To whom correspondence should be addressed (e-mail: eolson@hamon.swmed.edu).

STIM1 signalling controls store-operated calcium entry required for development and contractile function in skeletal muscle

It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.

Remodeling muscles with calcineurin

Ca2+ signaling plays a central role in hypertrophic growth of cardiac and skeletal muscle in response to mechanical load and a variety of signals. However, the mechanisms whereby alterations in Ca2+ in the cytoplasm activate the hypertrophic response and result in longterm changes in muscle gene expression are unclear. The Ca2+, calmodulin‐dependent protein phosphatase calcineurin has been proposed to control cardiac and skeletal muscle hypertrophy by acting as a Ca2+ sensor that couples prolonged changes in Ca2+ levels to reprogramming of muscle gene expression. Calcineurin also controls the contractile and metabolic properties of skeletal muscle by activating the slow muscle fiber‐specific gene program, which is dependent on Ca2+ signaling. Transcription factors of the NFAT and MEF2 families serve as endpoints for the signaling pathways whereby calcineurin controls muscle hypertrophy and fiber‐type. We consider these findings in the context of a model for Ca2+‐regulated gene expression in muscle cells and discuss potential implications of these findings for pharmacologic modification of cardiac and skeletal muscle function. BioEssays 22:510–519, 2000.

Physiological and psychological variables predict compliance to prescribed exercise therapy in patients recovering from myocardial infarction.

&NA; Previous research has documented high rates of noncompliance to prescribed medical therapy in patients recovering from myocardial infarction (MI). This study was undertaken to determine if patients who subsequently drop out of a structured cardiac rehabilitation program could be prospectively distinguished from those who remain in the program based upon their initial baseline characteristics. Thirty‐five consecutive patients with recent MIs underwent comprehensive physical and psychological assessments at entry into the program, and were followed for a period of 1 year. The 14 patients who dropped out of the program could be distinguished from the compliers on the basis of their reduced left ejection fraction assessed by first pass radionuclide angiography at rest and during peak exercise. In addition, their psychological profiles assessed by the MMPI indicated the dropouts were more depressed, hypochondriacal, anxious, and introverted and had lower ego strength than those who remained in the program. Statistical analysis further indicated that psychological variables were associated with noncompliance independently of physical status. These findings suggest that MI patients who are unlikely to adhere to this form of medical therapy may be prospectively identified based upon their initial physical and psychological characteristics.

Exercise responses before and after physical conditioning in patients with severely depressed left ventricular function

The ability of patients with severely impaired left ventricle function to perform short-term exercise and to participate in a cardiac rehabilitation program and attain physical training effects was evaluated. Treadmill exercise tests were performed before and after physical conditioning in 10 patients with a prior myocardial infarction and a left ventricular ejection fraction at rest of less than 27 percent (range 13 to 26) determined by radionuclide angiography. All patients participated in a supervised exercise program with a follow-up period of 4 to 37 (mean 12.7) months. Baseline exercise capacity showed marked variability, ranging from 4.5 to 9.4 (mean 7.0 +/- 1.9) METS, and improved to 5.5 to 14 (mean 8.5 +/- 2.9) METS after conditioning (p = 0.05). The oxygen pulse (maximal oxygen uptake/maximal heart rate) before and after conditioning was used to assess a training effect and increased significantly from 12.8 +/- 2.0 to 15.7 +/- 3.2 ml/beta (p less than 0.01). There was no exercise-related morbidity or mortality, although two patients died during the study period. It is concluded that selected patients with severely imparied left ventricular function can safely participate in a conditioning program and achieve cardiovascular training effects.

The Role of Modulatory Calcineurin-Interacting Proteins in Calcineurin Signaling

Modulatory calcineurin-interacting proteins (MCIPs), also known as the Down syndrome critical region 1 (DSCR1) and DSCR1-like proteins, are a recently described family of small, structurally related proteins that are preferentially expressed in heart, skeletal muscle, and brain. MCIP proteins can bind to and inhibit calcineurin, a calcium/calmodulin-regulated serine/threonine protein phosphatase that is activated during cardiac hypertrophy and failure. Transcription of the mammalian MCIP1 gene is induced by calcineurin, suggesting that it functions as an endogenous feedback regulator of calcineurin signal transduction. Forced expression of human MCIP1 protein in the hearts of transgenic mice attenuates the hypertrophic response to a broad range of stimuli. This review summarizes work from a number of laboratories on the structure, regulation, and function of MCIP proteins.

Multiple Domains of MCIP1 Contribute to Inhibition of Calcineurin Activity

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. Calcineurin-dependent signals are transduced to the nucleus by nuclear factor of activated T-cells (NFAT) transcription factors that undergo nuclear translocation upon dephosphorylation and promote transcriptional activation of target genes. Several endogenous proteins are capable of inhibiting the catalytic activity of calcineurin. Modulatorycalcineurin interacting protein 1 (MCIP1) is unique among these proteins on the basis of its pattern of expression and its function in a negative feedback loop to regulate calcineurin activity. Here we show that MCIP1 can be phosphorylated by MAPK and glycogen synthase kinase-3 and that phosphorylated MCIP1 is a substrate for calcineurin. Peptides corresponding to the substrate domain competitively inhibit calcineurin activity in vitro. However, a detailed structure/function analysis of MCIP1 reveals that either of two additional domains of MCIP1 is sufficient for binding to calcineurin in vitro and for inhibition of calcineurin activity in vivo. We conclude that MCIP1 inhibits calcineurin through mechanisms that include, but are not limited to, competition with other substrates such as nuclear factor of activated T-cells.