Ming Qi

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes.

Left ventricular hypertrophy (approximately 40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes were isolated for functional studies. In myocytes, the effects of banding on intracellular Ca handling, contraction, and excitation-contraction (E-C) coupling were determined using indo 1 fluorescence and whole cell voltage clamp. After steady-state field or voltage-clamp stimulation to load the sarcoplasmic reticulum (SR), SR Ca content assessed by caffeine-induced Ca transients was the same in sham and banded groups. Despite this, cell shortening amplitudes were significantly depressed in the banded group, suggesting altered contractile properties. In banded rats, the SR Ca-adenosinetriphosphatase (Ca-ATPase) mRNA level was reduced, as was homogenate thapsigargin-sensitive SR Ca-ATPase, but cytosolic free Ca concentration ([Ca]i) decline attributed to SR Ca-ATPase activity in intact cells was not slowed. Banding also reduced Na/Ca exchange mRNA level but did not affect either Na-dependent sarcolemmal 45Ca transport in homogenate or the rate of [Ca]i decline in intact cells attributed to Na/Ca exchange (during caffeine-induced contractures). Banding also did not change the rate of [Ca]i decline mediated by the combined function of the mitochondrial Ca uptake and sarcolemmal Ca-ATPase in intact cells. Ca current (ICa) density and voltage dependence were the same in sham and banded groups. Ryanodine receptor mRNA, protein content, and ryanodine affinity were also unchanged in the banded group. At 1 mM extracellular Ca concentration ([Ca]o), banding did not affect E-C coupling efficacy in intact cells under voltage clamp (i.e., same contraction for given ICa and SR Ca load). However, when [Ca]o was reduced to 0.5 mM, the efficacy of E-C coupling was greatly depressed in the banded group (even though ICa and SR Ca content were matched). In summary, unloaded myocyte contraction was depressed in these hypertrophic hearts, but Ca transport was little altered, at 1 mM [Ca]o. However, reduction of [Ca]o to 0.5 mM appears to unmask a depressed fractional SR Ca release in response to a given ICa trigger and SR Ca load.

Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells.

We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously beating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells, VA). Intracellular calcium (Cai) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Nao), and (3) twitches induced by brief depolarizations with high [K]o solution (K-twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t1/2 for [Ca]i decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (approximately 20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t1/2 of CafC when Na-CaX was the main mechanism responsible for [Ca]i decline (t1/2 approximately 1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca]i decline during K-twitches (control t1/2, 0.84 +/- 0.05 seconds; VA t1/2, 0.48 +/- 0.06 second; P < .001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca]i reached during CafC used to assess maximal SR Ca content (427 +/- 49 nmol/L in control versus 665 +/- 75 nmol/L in VA cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Contraction-Dependent Hypertrophy of Neonatal Rat Ventricular Myocytes: Potential Role for Focal Adhesion Kinase

Focal adhesion kinase (FAK) and other protein tyrosine kinases (PTKs) found in focal adhesions regulate proliferation and cytoskeletal assembly of nonmuscle cells, but their role in hypertrophic growth of cardiac myocytes has not been investigated. Serum-free, primary cultures of spontaneously contracting and contractile-arrested neonatal rat ventricular myocytes (NRVMs) were used to determine the role of FAK and other PTKs in regulating cardiac gene expression and protein turnover associated with the hypertrophic phenotype. FAK was readily detected in focal adhesions and costameres of spontaneously contracting, hypertrophied myocytes, but was reduced in contractile-arrested cells. Chronic treatment of NRVMs with genistein (50µM), a relatively specific PTK inhibitor, prevented NRVM growth, as demonstrated by significant reductions (by 10–35%) in total protein, total protein/DNA ratio, and myofibrillar protein content. Genistein also significandy reduced myosin heavy chain (MHC) and actin synthesis, and increased MHC and actin degradation. Daidzein (50 µM), a weakly active analogue, had much less of an effect. Genistein also markedly reduced β-myosin heavy chain (β-MHC) and to a lesser extent atrial natriuretic factor (ANF) gene expression, thus reproducing many of the phenotypic features of cardiac myocyte atrophy produced by contractile arrest. Transient transfection of NRVMs with an expression vector containing the full-length coding sequence of chick FAK along with rat β-MHC and ANF promoter-luciferase constructs resulted in a 2–4-fold increase in luciferase activity, indicating that FAK stimulated transcription of fetal genes associated with the hypertrophic phenotype. Thus, FAK and/or other PTKs found in focal adhesions may play a role in both the transcriptional and posttranslational regulation of cardiac myocyte hypertrophy.