Nuclear Targeting of Akt Enhances Ventricular Function and Myocyte Contractility

Abstract

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...

Adenylyl Cyclase Type VI Increases Akt Activity and Phospholamban Phosphorylation in Cardiac Myocytes

“…Whether this represents nuclear translocation or, alternatively, increased activation of Akt already present in the nucleus was not determined. Nuclear-targeted Akt expression was found to increase cardiac PLB phosphorylation in transgenic mice (40), so nuclear Akt activation, seen after AC VI gene transfer in the present study, is likely to be an important mechanism in PLB phosphorylation.…”

“…Mice with cardiac-directed expression of a constitutively active Akt show cardiac hypertrophy and increased systolic function (52), but sustained expression of Akt can lead to heart failure (53). In contrast, cardiac-directed and nuclear-targeted Akt expression increases contractile function (40,54), inhibits apoptosis, and protects the heart during ischemia-reperfusion injury (55). In the present studies, we found nuclear translocation of activated Akt evoked by increases in AC VI levels.…”

“…For example, ␤AR stimulation, through PKA activation, phosphorylates PLB, which disinhibits sarcoplasmic reticulum Ca 2ϩ ATPase and facilitates Ca 2ϩ uptake into the sarcoplasmic reticulum, increasing cardiac function (57). However, sustained ␤AR activation is associated with cardiac myocyte apoptosis (58,59), which is not seen in animals expressing nuclear Akt (40) or AC VI (56). Reduction of PLB through antisense suppression or genetic ablation increases sarcoplasmic reticulum calcium cycling and increases cardiac function (60,61), and cardiac-directed expression of PLB decreases heart function (62).…”

Reduction in Kv Current Enhances the Temporal Dispersion of the Action Potential in Diabetic Myocytes: Insights From a Novel Repolarization Algorithm

“…Isolated LV myocytes were placed in a bath on the stage of an IX51, IX53, or IX71 (Olympus, Tokyo, Japan) microscopes for patch‐clamp measurements 19, 25, 30, 31. Experiments were conducted at 37°C.…”

“…Electrical signals were digitized using 250‐kHz 16‐bit resolution A/D converters (Digidata 1322, 1440A, and 1550; Molecular Devices) and recorded using pCLAMP 9.0 and 10 software (Molecular Devices) with low‐pass filtering at 2 kHz. Membrane capacitance (C m ) was measured in voltage‐clamp mode using a 5‐mV voltage step and pCLAMP software algorithm; this parameter was employed to normalize transmembrane currents 19, 20, 25, 30. Pipettes were pulled by means of a vertical (PB‐7; Narishige International Inc., East Meadow, NY), or horizontal (P‐1000; Sutter Instrument Co, Novato, CA) glass microelectrode pullers.…”