Abstract-Cytoplasmic overexpression of Akt in the heart results in a myopathy characterized by organ and myocyte hypertrophy. Conversely, nuclear-targeted Akt does not lead to cardiac hypertrophy, but the cellular basis of this distinct heart phenotype remains to be determined. Similarly, whether nuclear-targeted Akt affects ventricular performance and mechanics, calcium metabolism, and electrical properties of myocytes is unknown. Moreover, whether the expression and state of phosphorylation of regulatory proteins implicated in calcium cycling and myocyte contractility are altered in nuclear-targeted Akt has not been established. We report that nuclear overexpression of Akt does not modify cardiac size and shape but results in an increased number of cardiomyocytes, which are smaller in volume. Additionally, the heart possesses enhanced systolic and diastolic function, which is paralleled by increased myocyte performance. Myocyte shortening and velocity of shortening and relengthening are increased in transgenic mice and are coupled with a more efficient reuptake of calcium by the sarcoplasmic reticulum (SR). This process increases calcium loading of the SR during relengthening. The enhanced SR function appears to be mediated by an increase in SR Ca 2ϩ -ATPase2a activity sustained by a higher degree of phosphorylation of phospholamban. This posttranslational modification was associated with an increase in phospho-protein kinase A and a decrease in protein phosphatase-1. Together, these observations provide a plausible biochemical mechanism for the potentiation of myocyte and ventricular function in Akt transgenic mice. Therefore, nuclear-targeted Akt in myocytes may have important implications for the diseased heart. Key Words: Akt Ⅲ myocyte mechanics Ⅲ myocyte size and number P rotein kinase B, also referred to as Akt, phosphorylates multiple cytoplasmic and nuclear substrates implicated in cell survival and growth of several organs including the heart. 1 Although myocyte survival and cellular hypertrophy may be viewed as important adaptations of the overloaded heart against the onset of ventricular decompensation, 2 the targeted expression of constitutively activated Akt to the myocardium has resulted in cardiac hypertrophy [3][4][5][6][7] and ventricular dysfunction. 6 In these cases, however, transgene activity was widespread throughout cardiomyocytes at nonphysiological levels, raising the possibility that the nuclear accumulation of Akt may retain the antiapoptotic effects of this serine-threonine kinase, without promoting organ hypertrophy and alterations in cardiac performance. In this regard, hearts of mice expressing nuclear-targeted Akt show no evidence of myopathy 8 in contrast to other cardiac-specific Akt transgenics created with constitutively activated kinase. Targeting of Akt to myocyte nuclei preserves cell viability through the phosphorylation of survival factors within the nucleus that interfere with apoptotic death signaling. 1,8 -11 Thus, whether Akt is expressed in the cytoplasm or in t...

Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release due to alteration of the action potential (excitation-contraction ABSTRACTAnoxia of the heart causes failure of contraction before any irreversible injury occurs; the mechanism by which anoxia blocks cardiac excitation-contraction coupling is unknown. Studies in whole muscle are confounded by heterogeneity; however, achieving the low oxygen tensions required to study anoxia in a single myocyte during electrophysiological recording has been a barrier in experimental design. Guided by calculations of oxygen transport, we developed a system to insulate myocytes in an open dish from oxygen by a laminar counterflowing argon column, permitting free access to the cell by microelectrodes while maintaining a P02 <0.02 torr (1 torr = 133 Pa). In the absence of glucose, the amplitude of stimulated contraction of anoxic ventricular myocytes fell to

A technique has been developed for isolating a high yield of Ca2+-tolerant rod-shaped myocytes from the right and left ventricles of cat myocardial tissue. Myocytes were prepared by retrograde perfusion of the coronary arteries via the aorta with a nominally Ca2+-free (20-30 microM) modified Krebs-Henseleit buffer containing 0.12% collagenase. After exposure to physiological levels of Ca2+ (1-2.5 mM), the cells retained rod-shaped morphology, exhibited clear cross striations, and excluded the dye trypan blue (0.4%). Initial percents of viable Ca2+-tolerant rod-shaped cells were 58.6 +/- 3.4 (SE) and 51.8 +/- 3.5 for right and left ventricular cells, respectively. Viability studies demonstrated that these values decreased approximately 10% at the conclusion of a 2-h incubation in 1 mM Ca2+. The total numbers of rod-shaped myocytes obtained were 4.48 X 10(7) and 3.89 X 10(7) in nominal (8-10 microM) and 1 mM Ca2+-containing buffer, respectively. A total of 3.44 +/- 0.40 X 10(6) rod-shaped Ca2+-tolerant myocytes was initially isolated per gram of tissue wet weight. Measurements of cell length, width, and sarcomere length demonstrated no significant differences between right and left ventricular cells suspended in nominal (8-10 microM) and 1 mM Ca2+-containing buffer. No significant difference was found in the percent of binucleate cells when right and left ventricular myocytes were compared. These results demonstrate that a stable population of Ca2+-tolerant myocytes with similar morphological characteristics can be isolated from the right and left ventricles of cat myocardium.

The incidence of pulmonary atelectasis following head and neck surgery is not well reported. This study retrospectively evaluated the incidence of pulmonary atelectasis in 161 head and neck cancer patients, with 152 being evaluable. There were 90 patients evaluated following pectoralis musculocutaneous flap reconstruction with their effective flap size and 71 nonflap patients as a control group. Clinical findings were correlated to radiographic scores. Of pectoralis musculocutaneous flap patients screened for preexisting pulmonary disease (PEPD), nine of 45 (20%) demonstrated pulmonary atelectasis in the first 24 hours compared with 10 of 39 or 25.6% nonflap controls. Major pulmonary atelectasis was not found in the pectoralis musculocutaneous flap patients by scoring criteria, and in only one of 39 (2.6%) nonflap patients. In flaps larger than 40 cm2, the incidence was eight of 37 (21.6%), with no major pulmonary atelectasis noted. Only one of nine (11.1%) patients with radiographic pulmonary atelectasis exhibited clinical symptoms (three of 10 or 30% control). In patients with PEPD and pectoralis musculocutaneous flaps, 22 of 45 (48.9%) had evidence of pulmonary atelectasis in contrast to 13 of 32 or 40.6% controls. There were two of 45 (4.4%) who had major pulmonary atelectasis with zero of 32 in the nonflap group. For flaps larger than 40 cm2, the incidence was 19 of 39 (48.7%) with two of 39 (5.1%) scored as major pulmonary atelectasis. The clinical correlation for this group and the major pulmonary atelectasis group was each approximately 50% compared to 15.4% for nonflap patients.(ABSTRACT TRUNCATED AT 250 WORDS)