Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.

Phosphorylation of Troponin I by Protein Kinase A Accelerates Relaxation and Crossbridge Cycle Kinetics in Mouse Ventricular Muscle

https://mcb.berkeley.edu/labs/zucker/PDFs/Pan_Neuron62,539%5BTOTAL%5D.pdf

A General Model of Synaptic Transmission and Short-Term Plasticity

1. We have studied exocytosis and endocytosis in the synaptic terminal of salamander rods using a combination of Ca2+ imaging, capacitance measurement and the photolysis of Ca2+ buffers. 2. The average cytoplasmic Ca2+ concentration at the dark resting potential was 2-4 microM. 3. An average cytoplasmic Ca2+ concentration of 2-4 microM maintained a high rate of continuous exocytosis and endocytosis. 4. Changes in the rate of exocytosis were followed in less than 0.7 s by compensatory changes in the rate of endocytosis. 5. Vesicle cycling in the rod synapse is specialized for graded transmission and differs from that previously described for synapses that release synchronized bursts of transmitter.

Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.

Brief increases in [Ca2+]i can result in prolonged changes in neuronal properties. A Ca2+-dependent modulation of the hyperpolarization-activated cation current (Ih) controls the slow recurrence of synchronized thalamocortical activity. Here we show that the persistent activation of Ih is initiated by rapidly increased [Ca2+]i and subsequent production of cAMP. The modulation is maintained via a facilitated interaction of cAMP with open (voltage-gated) h-channels, inducing prolonged activation of Ih that may outlast the presence of increased free [Ca2+]i and [cAMP]i. This persistent Ih activation may control the presence and periodicity of both normal and abnormal synchronized thalamocortical rhythms.

Modulation of a pacemaker current through Ca(2+)-induced stimulation of cAMP production.

The effects of ventricular myosin heavy chain (MHC) composition on the kinetics of activation and relaxation were examined in both chemically skinned and intact myocardial preparations from adult rats. Thyroid deficiency was induced to alter ventricular MHC isoform expression from ∼80 %α-MHC/20 %β-MHC in euthyroid rats to 100 %β-MHC, without altering the expression of thin-filament-associated regulatory proteins.


In single skinned myocytes, increased expression of β-MHC did not significantly affect either maximal Ca2+-activated tension (P0) or the Ca2+ sensitivity of tension (pCa50). However, unloaded shortening velocity (V0) decreased by 80 % due to increased β-MHC expression.


The kinetics of activation and relaxation were examined in skinned multicellular preparations using the caged Ca2+ compound DM-nitrophen and caged Ca2+ chelator diazo-2, respectively. Myocardium expressing 100 %β-MHC exhibited apparent rates of submaximal and maximal tension development (kCa) that were 60 % lower than in control myocardium, and a 2-fold increase in the half-time for relaxation from steady-state submaximal force.


The time courses of cell shortening and intracellular Ca2+ transients were assessed in living, electrically paced myocytes, both with and without β-adrenergic stimulation (70 nm isoproterenol (isoprenaline)). Thyroid deficiency had no affect on either the extent of myocyte shortening or the resting or peak fura-2 fluorescence ratios. However, induction of β-MHC expression by thyroid deficiency was associated with increased half-times for myocyte shortening and relengthening and increased half-time for the decay of the fura-2 fluorescence ratio. Qualitatively similar results were obtained in both the absence and the presence of β-adrenergic stimulation although the β-agonist accelerated the kinetics of the twitch and the Ca2+ transient.


Collectively, these data provide evidence that increased β-MHC expression contributes significantly to the observed depression of contractile function in thyroid deficient myocardium by slowing the rates of both force development and force relaxation.







In striated muscle, Ca2+ binding to troponin C (TnC) initiates a series of events that permit strong interaction between myosin and actin (contraction), while dissociation of Ca2+ from TnC leads to reversal of these events and detachment of myosin from actin (relaxation). While Ca2+ binding to TnC initiates contraction, complete activation of the thin filament in terms of tension and the kinetics of tension development most probably involves the synergistic actions of Ca2+ and strong-binding myosin cross-bridges (Swartz & Moss, 1992; Geeves & Lehrer, 1994; Swartz et al. 1996). Furthermore, the kinetics of interaction of myosin with actin are thought to be determined at least in part by the myosin heavy chain (MHC) content of a given muscle. For example, the maximal rate of tension redevelopment is 8-fold faster in fast-twitch skeletal muscle fibres expressing type II B MHC than in slow-twitch fibres expressing type I MHC, i.e. cardiac β-MHC (Metzger & Moss, 1990). Also, muscle-to-muscle variations in maximal shortening velocity, an index of cross-bridge detachment rate, are thought to be determined by MHC content (Harris et al. 1994; VanBuren et al. 1995; Schiaffino & Reggiani, 1996). To date, little information is available as to whether there is MHC isoform-specific modulation of the kinetics of force development or relaxation, especially in heart muscle.

Two cardiac MHC isoforms, α and β, have been identified in the adult mammalian ventricle and are products of two closely related genes (Lompre et al. 1984). The phenotypic expression of cardiac MHC isoforms is dynamic and subject to a number of physiological influences, including regulation by hormones such as thyroid hormone (Lompre et al. 1984). Thyroid deficiency results in a complete remodelling of ventricular MHC distribution from ∼80 %α-MHC/20 %β-MHC to 100 %β-MHC (Morkin, 1993). It is interesting to note that while cardiac MHC expression varies with thyroid state, altered levels of triiodothyronine (T3) do not appear to alter the phenotypic expression of other myofibrillar protein isoforms, such as the thin filament proteins troponin I (TnI) and troponin T (TnT) (Averyhart-Fullard et al. 1994; Akella et al. 1997). However, it is well known that thyroid state markedly affects the Ca2+ handling properties of the sarcoplasmic reticulum (SR) (Kiss et al. 1994). Therefore, studies examining modulation of myocardial contraction subsequent to altered thyroid status must account for changes in Ca2+ handling as well as myofilament effects mediated by increased expression of β-MHC.

Here, we investigated the influence of thyroid status and altered MHC protein expression on the kinetics of myocardial contraction in both chemically skinned and intact myocardial preparations. The relative proportions of ventricular MHC isoforms in adult rat myocardium were transformed from predominantly α-MHC to exclusively β-MHC by inducing a thyroid deficient state. In skinned myocardium, activation and relaxation kinetics were examined using the photolabile caged Ca2+ compound DM-nitrophen (Kaplan & Ellis-Davies, 1988) and caged Ca2+ chelator diazo-2 (Adams et al. 1989), respectively. Electrically stimulated intact myocytes were used to examine possible interactions between thyroid deficiency-induced changes in MHC content and Ca2+ handling properties of the SR in determining twitch characteristics.

R. Y. Tsien

Papers

Biologically Useful Chelators That Take Up Ca2+ Upon Illumination.