YeQing Pi

Cardiac Troponin I Gene Knockout A Mouse Model of Myocardial Troponin I Deficiency

Troponin I is a subunit of the thin filament-associated troponin-tropomyosin complex involved in calcium regulation of skeletal and cardiac muscle contraction. We deleted the cardiac isoform of troponin I by using gene targeting in murine embryonic stem cells to determine the developmental and physiological effects of the absence of this regulatory protein. Mice lacking cardiac troponin I were born healthy, with normal heart and body weight, because a fetal troponin I isoform (identical to slow skeletal troponin I) compensated for the absence of cardiac troponin I. Compensation was only temporary, however, as 15 days after birth slow skeletal troponin I expression began a steady decline, giving rise to a troponin I deficiency. Mice died of acute heart failure on day 18, demonstrating that some form of troponin I is required for normal cardiac function and survival. Ventricular myocytes isolated from these troponin I-depleted hearts displayed shortened sarcomeres and elevated resting tension measured under relaxing conditions and had a reduced myofilament Ca sensitivity under activating conditions. The results show that (1) developmental downregulation of slow skeletal troponin I occurs even in the absence of cardiac troponin I and (2) the resultant troponin I depletion alters specific mechanical properties of myocardium and can lead to a lethal form of acute heart failure.

Phosphorylation of Troponin I Controls Cardiac Twitch Dynamics: Evidence From Phosphorylation Site Mutants Expressed on a Troponin I-Null Background in Mice

The cardiac myofilament protein troponin I (cTnI) is phosphorylated by protein kinase C (PKC), a family of serine/threonine kinases activated within heart muscle by a variety of agonists. cTnI is also a substrate for cAMP-dependent protein kinase (PKA) activated during &bgr;-adrenergic signaling. To investigate the role of cTnI phosphorylation in contractile regulation by these pathways, we generated transgenic mice harboring a mutated cTnI protein lacking phosphorylation sites for PKC (serine43/45 and threonine144 mutated to alanine) and for PKA (serine23/24 mutated to alanine). Transgenic mice were interbred with cTnI-knockout mice to ensure the absence of endogenous phosphorylatable cTnI. Here, we report that regulation of myocyte twitch kinetics by &bgr;-stimulation and by endothelin-1 was altered in myocytes containing mutant cTnI. In wild-type myocytes, the &bgr;-agonist isoproterenol decreased twitch duration and relaxation time constant (&tgr;) by 37% to 44%. These lusitropic effects of isoproterenol were reduced by about half in nonphosphorylatable cTnI mutant myocytes and were absent in cTnI mutants also lacking phospholamban (generated by crossing cTnI mutants with phospholamban-knockout mice). These observations are consistent with important roles for both cTnI and phospholamban phosphorylation in accelerating relaxation after &bgr;-adrenergic stimulation. In contrast, endothelin-1 increased twitch duration by 32% and increased &tgr; by 58%. These endothelin-1 effects were substantially blunted in nonphosphorylatable cTnI myocytes, indicating that PKC phosphorylation of cTnI slows cardiac relaxation and increases twitch duration. We propose that &bgr;-agonists and endothelin-1 regulate cardiac twitch dynamics in opposite directions in part through phosphorylation of the myofilament protein cTnI on distinct sites.

Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium

Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin‐tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol‐12‐myristate‐13‐acetate (PMA) or endothelin‐1 (ET‐1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca2+ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca2+ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines43/45 and threonine144 (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca2+ sensitivity required the presence of phosphorylatable cTnI. For comparison, the β‐adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca2+ sensitivity. These β‐adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca2+ and ATP, thereby promoting efficient ATP utilisation during contraction.

Endothelin-1 and photoreleased diacylglycerol increase L-type Ca2+ current by activation of protein kinase C in rat ventricular myocytes

1 The amphotericin B‐perforated whole‐cell patch clamp technique was used to determine the modulation of L‐type Ca2+ channels by protein kinase C (PKC)‐mediated pathways in adult rat ventricular myocytes. 2 Application of 10 nM endothelin‐1 (ET‐1) increased peak Ca2+ current (ICa) by 28.2 ± 2.5 % (n= 13) and slowed current decay. These effects were prevented by the endothelin receptor antagonist PD145065 (10 μM) and by the PKC inhibitor chelerythrine (8 μM). 3 To establish if direct activation of PKC mimicked the ET‐1 effect, the active and inactive phorbol esters (phorbol‐12‐myristate‐13‐acetate and 4α‐phorbol‐12, 13‐didecanoate) were tested. Both phorbol esters (100 nM) resulted in a small (∼10 %) increase in ICa, suggesting PKC‐independent effects. 4 Bath application of dioctanoylglycerol (diC8), a diacylglycerol (DAG) analogue which is capable of directly activating PKC, caused a gradual decline in peak ICa (50.4 ± 6.2 %, n= 5) and increased the rate of current decay. These effects were unaffected by the PKC inhibitor chelerythrine (8 μM). 5 Intracellular photorelease of caged diC8 with 3 or 10 s exposure to UV light produced a concentration‐dependent increase in peak ICa (20.7 ± 8.5 % (n= 8) for 3 s UV and 60.8 ± 11.4 % (n= 13) for 10 s UV), which could be inhibited by chelerythrine. 6 Our results demonstrate that both ET‐1 and intracellularly photoreleased diC8 increase ICa by a PKC‐mediated pathway, which is in direct contrast to the PKC‐independent inhibition of ICa produced by bath‐applied diC8. We conclude that specific cellular pools of DAG are crucially important in the regulation of ICa by PKC.

Localization of Functional Endothelin Receptor Signaling Complexes in Cardiac Transverse Tubules

Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ETA receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-β1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-ϵ-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte “surface” mimicking the negative inotropic effects of bath-applied PKC activators and “deep” release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such “detubulated” cardiac myocytes showed no positive inotropic response to 20 nm ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-β1-PKC-ϵ signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.

Positive inotropy mediated by diacylglycerol in rat ventricular myocytes

Many neurohormones stimulate phospholipid hydrolysis and elevate diacylglycerol in the mammalian heart, but the physiological consequences of these intracellular events are unclear. Regulation of myocardial contraction by diacylglycerol was investigated in the present study by releasing the diacylglycerol analogue dioctanoylglycerol (diC8) within adult rat ventricular myocytes by using a light-sensitive caged compound. This approach permitted us to avoid exposure of myocytes to extracellular diC8 and yet to control the amount of diC8 released into the cells. Photorelease of diC8 produced a slowly developing (half-time, 1.9 +/- 0.1 minute; n = 26) but robust (406 +/- 42%) enhancement of twitch amplitude in electrically paced myocytes (0.5 Hz, 1 mmol/L Ca2+, Ringers solution [pH 7.4], 22 degrees C). This positive inotropic effect was dose dependent, stereospecific for the S-enantiomer of diC8, synergistically enhanced by arachidonic acid, and blocked by the protein kinase C inhibitor chelerythrine. The data provide evidence that diacylglycerol can induce a strong positive inotropic effect in mammalian ventricular muscle, possibly by activating protein kinase C. By contrast, perfusion of diC8 extracellularly onto myocytes caused a 42 +/- 2% decline in twitch amplitude, in accordance with previous reports. To account for this dependence on how diC8 is applied, we postulate that diC8 has distinct physiological actions at intracellular and extracellular sites. The peptide neurohormone endothelin-1, which elevates diacylglycerol in cardiac tissues, produced a positive inotropic effect that was similar to the response to photoreleased diC8. The diacylglycerol/protein kinase C pathway has now become a good candidate for mediator of at least a component of the positive inotropy associated with agents that stimulate phospholipid turnover in adult mammalian myocardium.

Role of intracellular Ca2+ and pH in positive inotropic response of cardiomyocytes to diacylglycerol.

Diacylglycerol has been hypothesized to mediate the positive inotropic response of myocardium to the α-adrenergic agonists angiotensin II and endothelin. The mechanism of action of diacylglycerol was examined here in adult rat ventricular myocytes by releasing dioctanoylglycerol (diC8) intracellularly from a caged compound while monitoring Ca2+ transients and pH with fluorescent indicators. DiC8caused a three- to fourfold increase in twitch amplitude and a twofold increase in the systolic Ca2+transient. No other parameter was consistently influenced by diC8, including the kinetics of Ca2+ cycling, the Ca2+ content of the sarcoplasmic reticulum, or the myofilament Ca2+sensitivity. DiC8 also had no detectable effect on intracellular pH or Na+/H+antiport activity. Consistent with this finding, the Na+/H+exchange inhibitor N-ethylisopropyl amiloride was without effect on the positive inotropic response to diC8. The marked enhancement of systolic Ca2+ by diC8 suggests that the process of excitation-contraction coupling is an important and possibly preferred target of diacylglycerol-protein kinase C signaling in myocardium.

Stereospecific protein kinase C activation by photolabile diglycerides

Abstract The synthesis and photochemistry of diglycerides designed to photolyze to bioactive diacylglycerols and optimized for facile incorporation into biological membranes is described. Stereospecific activation of protein kinase C in vitro and in living cells is demonstrated.