Background-Mutations in the ␥ 2 subunit (PRKAG2) of AMP-activated protein kinase produce an unusual human cardiomyopathy characterized by ventricular hypertrophy and electrophysiological abnormalities: Wolff-ParkinsonWhite syndrome (WPW) and progressive degenerative conduction system disease. Pathological examinations of affected human hearts reveal vacuoles containing amylopectin, a glycogen-related substance. Methods and Results-To elucidate the mechanism by which PRKAG2 mutations produce hypertrophy with electrophysiological abnormalities, we constructed transgenic mice overexpressing the PRKAG2 cDNA with or without a missense N488I human mutation. Transgenic mutant mice showed elevated AMP-activated protein kinase activity, accumulated large amounts of cardiac glycogen (30-fold above normal), developed dramatic left ventricular hypertrophy, and exhibited ventricular preexcitation and sinus node dysfunction. Electrophysiological testing demonstrated alternative atrioventricular conduction pathways consistent with WPW. Cardiac histopathology revealed that the annulus fibrosis, which normally insulates the ventricles from inappropriate excitation by the atria, was disrupted by glycogen-filled myocytes. These anomalous microscopic atrioventricular connections, rather than morphologically distinct bypass tracts, appeared to provide the anatomic substrate for ventricular preexcitation. Conclusions-Our data establish PRKAG2 mutations as a glycogen storage cardiomyopathy, provide an anatomic explanation for electrophysiological findings, and implicate disruption of the annulus fibrosis by glycogen-engorged myocytes as the cause of preexcitation in Pompe, Danon, and other glycogen storage diseases.

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 mol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the springlike domain of titin. Doxorubicin treatment for 1 h resulted in ϳ3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.

Neuregulin (NRG)-1␤ has a prosurvival effect on cardiac myocytes via the phosphatidylinositol-3-kinase/Akt pathway, but the physiological regulators of this system in the intact heart are unknown. In this study, we tested the hypothesis that reactive oxygen species regulate NRG/erbB signaling. We used isolated adult rat ventricular myocytes (ARVMs) or cardiac microvascular endothelial cells (CMECs) in monoculture, or together in coculture. H 2 O 2 induced NRG-1␤ release from CMECs in a concentration-dependent manner, and conditioned medium from H 2 O 2 -treated CMEC activated ARVM erbB4. NRG-1␤ release occurred via proteolytic cleavage of 115-kDa transmembrane NRG-1␤ and was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline. In myocyte monoculture, H 2 O 2 induced erbB4-dependent, but NRG-independent, activation of Akt. To elucidate the bioactivity of CMEC-derived NRG-1␤ on ARVMs, we examined H 2 O 2 -induced myocyte apoptosis in co-culture using an antibody to NRG-1␤. The percentages of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly higher in the anti-NRG-1␤ group than in the control group. The change in apoptosis induced by anti-NRG-1␤ in co-culture was similar in magnitude to the protection of myocytes by addition of recombinant NRG-1␤ to ARVM monocultures. Activation of NRG/erbB paracrine signaling was also seen in the intact heart subjected to oxidative stress by ischemia-reperfusion injury. Isolated perfused mouse hearts subjected to 15 min of ischemia, followed by 30 min of reperfusion, showed complete proteolytic cleavage of 115-kDa NRG-1␤, with concomitant erbB4 phosphorylation. These results demonstrate that reactive oxygen species activate NRG-1␤/erbB4 paracrine signaling in the heart and suggest that this system is involved in cardiac adaptation to oxidative stress. Neuregulin-1 (NRG)1 /erbB signaling is essential for cardiac development (1-5). Moreover, cardiotoxicity of trastuzumab (Herceptin), an erbB2-targeted antibody used in treatment of erbB2-positive cancer, suggests that this pathway also plays a role in the regulation of cardiac structure and function in the postnatal heart (6). Cardiac microvascular endothelial cells (CMECs) express NRG (7), and recombinant NRG-1␤ protects myocytes against anthracycline-and ␤-adrenergic receptor-induced cell injury and death (8 -10). This prosurvival effect of NRG-1␤ occurs through the activation of erbB2 and erbB4 receptor tyrosine kinases in myocytes with downstream signaling in the phosphatidylinositol-3-kinase/Akt pathway. The in vivo role of erbB signaling in cardioprotection is further supported by the finding that mice with a cardiac-specific erbB2 conditional knock-out survive to birth but have accelerated heart failure after injury by pressure-overload or Adriamycin (11, 12). These observations suggest that the NRG/erbB pathway acts as a stress-responsive signal between CMECs and myocytes to maintain cell survival and cardiac function.We have cloned eight isoforms of NRG-1␤ fr...

Primary familial brain calcification (PFBC) is a genetically heterogeneous disorder characterized by bilateral calcifications in the basal ganglia and other brain regions. The genetic basis of this disorder remains unknown in a significant portion of familial cases. Here, we reported a recessive causal gene, MYORG, for PFBC. Compound heterozygous or homozygous mutations of MYORG co-segregated completely with PFBC in six families, with logarithm of odds (LOD) score of 4.91 at the zero recombination fraction. In mice, Myorg mRNA was expressed specifically in S100β-positive astrocytes, and knockout of Myorg induced the formation of brain calcification at 9 months of age. Our findings provide strong evidence that loss-of-function mutations of MYORG cause brain calcification in humans and mice.

Cardiac myocyte erbB2 expression is required for maintenance of normal cardiac structure and function, though its role in cardiac cellular physiology is incompletely understood. We tested the hypothesis that erbB2 signaling modulates focal adhesion formation via activation of a src/FAK pathway using adult rat ventricular myocytes in primary culture. The erbB ligand neuregulin-1β (NRG-1β) induced phosphorylation of Src at Y416 and Y215, and FAK at Y861. Using antibody and pharmacological inhibitor strategies, we found that FAK activation was erbB2-and Srcdependent, but independent of PI3-kinase/Akt pathway. Furthermore, NRG-1β stimulated the formation of a multiprotein complex between erbB2, FAK, p130 CAS and paxillin within 30 min, and induced lamellipodia with longitudinal elongation of the myocytes within days. The extension of lamellipodia resulted in restoration of cell-to-cell contact between isolated myocytes, allowing for synchronous beating. These effects of NRG-1β were prevented by a src inhibitor as well as an antibody to erbB2. These results suggest the potential role of NRG-1β/erbB2/Src/FAK signaling in the maintenance and repair of electrical and mechanical coupling in cardiomyocytes.

Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with ϳ10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of ␤-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge.The physiological mechanisms by which cells dynamically shift between steady states of tissue function, for example from tissue growth to cell survival during periods of metabolic stress, are incompletely understood. The activity of large sets of proteins must be coordinately regulated during these shifts. Many of the proteins involved in cell growth are stabilized by the constitutively expressed chaperone heat shock protein (Hsp) 1 90 (1, 2). Dissociation of Hsp90 from its client proteins induced by Hsp90 inhibitors leads to rapid degradation of proteins involved in cell growth and the activation of a stress response (3). Thus, pharmacologically induced disruption in Hsp90 chaperone function alone can cause a cell to "shift gears" to a stress-mode.Hsp90 inhibitors such as geldanamycin (GA) bind with high affinity to a conserved pocket in the Hsp90 family of proteins and thereby prevent the ATP binding that is required for chaperone function (4). The relative affinity of Hsp90 for GA, ADP, and ATP were recently measured and exhibited dissociation constants of 0.2, 12, and 124 M, respectively (5). The relatively high affinity of Hsp90 for GA is consistent with its established potency as an inhibitor of Hsp90 chaperone function. The ϳ10-fold higher affinity of Hsp90 for ADP versus ATP is intriguing. Under basal conditions, when the intracellular [ATP]/[ADP]ratio is between 2 and 10:1 (6), the higher affinity of Hsp90 for ADP suggests that a small drop in the ATP/ADP ratio will result in a relatively large drop in the proportion of Hsp90 occupied by ATP and, therefore, available for client protein stabilization.These observations led us to hypothesize that Hsp90 ...

The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to beta-adrenergic receptor (beta-AR)-stimulated apoptosis, and neuregulin-1beta (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to beta-AR-stimulated JNK phosphorylation and cell death with 0.1 microM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 microM NE in quiescent cells. NRG suppressed beta-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.

Nitric oxide (NO)-induced apoptosis in neurons is an important cause of neurodegenerative disease in humans. The cold-inducible protein RBM3 mediates the protective effects of cooling on apoptosis induced by various insults. However, whether RBM3 protects neural cells from NO-induced apoptosis is unclear. This study aimed to investigate the neuroprotective effect of RBM3 on NO-induced apoptosis in human SH-SY5Y neuroblastoma cells. Firstly, we demonstrated that mild hypothermia (32 °C) induces RBM3 expression and confers a potent neuroprotective effect on NO-induced apoptosis, which was substantially diminished when RBM3 was silenced by siRNA. Moreover, overexpression of RBM3 exhibited a strong protective effect against NO-induced apoptosis. Signaling pathway screening demonstrated that only p38 inhibition by RBM3 provided neuroprotective effect, although RBM3 overexpression could affect the activation of p38, JNK, ERK, and AKT signaling in response to NO stimuli. Notably, RBM3 overexpression also blocked the activation of p38 signaling induced by transforming growth factor-β1. Furthermore, both RBM3 overexpression and mild hypothermia abolished the induction of miR-143 by NO, which was shown to mediate the cytotoxicity of NO in a p38-dependent way. These findings suggest that RBM3 protects neuroblastoma cells from NO-induced apoptosis by suppressing p38 signaling, which mediates apoptosis through miR-143 induction.