Background:The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. Methods: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. Results: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were deter-

Abstract-Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT 1 ) and type 2 (AT 2 ) receptor-expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT 1 and AT 2 receptor subtypes. 4 and its function is less well delineated. Normally present only at low levels in the adult myocardium, AT 2 expression is upregulated in the pathological remodeling heart. 3 AT 2 -mediated cardiovascular responses have been characterized as generally opposing AT 1 -mediated growth effects, but some studies indicate that the AT 2 receptor is also involved in inducing left ventricular hypertrophy. 5,6 We have also shown that the AT 2 receptor can mediate myocyte hypertrophy independent of Ang II in isolated neonatal cardiomyocytes. 7 Thus, the role of the AT 2 receptor remains ambiguous.Autophagy is an intracellular degradation process that involves the destruction of long-lived proteins and organelles. Recently, autophagy has been particularly recognized as important in the turnover of cytoplasmic constituents in the heart. Autophagic activity is commonly increased in the heart under conditions that also involve upregulation of the reninangiotensin system. For example, high levels of autophagy are found in the heart after acute and chronic ischemia, heart failure, and neonatal starvation. 8 There is emerging evidence that pressure overload, a major risk factor for cardiac hypertrophy and heart failure, is associated with an excess of autophagy, which is ultimately maladaptive under such conditions of hemodynamic stress. 9 The proximal triggers for autophagy in the heart are not known.AT 1 receptor activation stimulates protein synthesis and protein turnover in cardiomyocytes. Intracellular protein aggregation is an important precursor event for cardiomyocyte autophagy. 10 Thus, links among activation of the renin-angiotensin system, protein turnover stimulation, and autophagic upregulation may be identified in several pathophysiological myocardial conditions. Therefore, in this study we hypothesized that the AT 1 and AT 2 receptor subtypes play a

The present study investigated the effects of gender on intracellular [Ca2+] ([Ca2+]i) in freshly isolated rat cardiac myocytes. Changes in [Ca2+]i in response to varied extracellular [Ca2+], different stimulus frequencies and addition of caffeine and isoprenaline were monitored using fura-2 in both male and female cardiac myocytes. Increasing extracellular [Ca2+] and stimulus frequency resulted in significant increases in peak [Ca2+] and the amplitude of the Ca2+ transient in both male and female cardiac myocytes. However, as extracellular [Ca2+] was raised, peak [Ca2+] and the amplitude of the Ca2+ transient increased significantly more in male than female cardiac myocytes. In addition a significant difference between male and female cells at each stimulus frequency was apparent. The time course of decay of the Ca2+ transient was significantly slower in female cardiac myocytes when compared with male cardiac myocytes, along with significantly slowed times to peak shortening and 50% relaxation, and a reduced extent of shortening. There was no significant difference in the amplitude of caffeine-induced [Ca2+]i responses between male and female cells, however, [Ca2+]i increased more readily in male cells than in female cells when isoprenaline was added. The data demonstrate that, under a variety of conditions, intracellular [Ca2+] rises to higher levels in cardiac myocytes from male as compared to female rats.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

Cardiac metabolic stress is a hallmark of many cardiac pathologies, including diabetes. Cardiac glycogen mis-handling is a frequent manifestation of various cardiopathologies. Diabetic females have a higher risk of heart disease than males, yet sex disparities in cardiac metabolic stress settings are not well understood. Oestrogen acts on key glycogen regulatory proteins. The goal of this study was to evaluate sex-specific metabolic stress-triggered cardiac glycogen handling responses. Male and female adult C57Bl/6J mice were fasted for 48h. Cardiac glycogen content, particle size, regulatory enzymes, signalling intermediates and autophagic processes were evaluated. Female hearts exhibited 51% lower basal glycogen content than males associated with lower AMP-activated-kinase (AMPK) activity (35% decrease in pAMPK:AMPK). With fasting, glycogen accumulated in female hearts linked with decreased particle size and upregulation of Akt and AMPK signalling, activation of glycogen synthase and inactivation of glycogen phosphorylase. Fasting did not alter glycogen content or regulatory proteins in male hearts. Expression of glycogen autophagy marker, starch-binding-protein-domain-1 (STBD1), was 63% lower in female hearts than males and increased by 69% with fasting in females only. Macro-autophagy markers, p62 and LC3BII:I ratio, increased with fasting in male and female hearts. This study identifies glycogen autophagy ('glycophagy') as a potentially important component of the response to cardiac metabolic stress. Glycogen autophagy occurs in association with a marked and selective accumulation of glycogen in the female myocardium. Our findings suggest that sex-specific differences in glycogen handling may have cardiopathologic consequences in various settings, including diabetic cardiomyopathy.