In vivo acceleration of heart relaxation performance by parvalbumin gene delivery.

Defective cardiac muscle relaxation plays a causal role in heart failure. Shown here is the new in vivo application of parvalbumin, a calcium-binding protein that facilitates ultrafast relaxation of specialized skeletal muscles. Parvalbumin is not naturally expressed in the heart. We show that parvalbumin gene transfer to the heart in vivo produces levels of parvalbumin characteristic of fast skeletal muscles, causes a physiologically relevant acceleration of heart relaxation performance in normal hearts, and enhances relaxation performance in an animal model of slowed cardiac muscle relaxation. Parvalbumin may offer the unique potential to correct defective relaxation in energetically compromised failing hearts because the relaxation-enhancement effect of parvalbumin arises from an ATP-independent mechanism. Additionally, parvalbumin gene transfer may provide a new therapeutic approach to correct cellular disturbances in calcium signaling pathways that cause abnormal growth or damage in the heart or other organs.

Effects of aging on single cardiac myocyte function in Fischer 344 x Brown Norway rats.

The Fischer 344 x Brown Norway (F344xBN) rat has been demonstrated to have a lower incidence of age-related pathology than other rat strains. Therefore, to elucidate the effects of aging on cardiac function, uncomplicated by compensatory effects caused by age-related pathology, cardiac myocytes were isolated from female F344xBN rats at 6 (young) and 32-33 (old) mo of age. Myocytes showed an increase in the relative amount of beta-myosin heavy chain with advanced age and a significant rightward shift in the tension-pCa curve from 5.78 +/- 0.02 pCa units in young adult myocytes to 5.66 +/- 0.03 pCa units. Consistent with a shift to a slower myosin isoform, the time from stimulation to peak sarcomere shortening increased with age from 50.5 +/- 1.3 to 58.9 +/- 1.0 ms. In contrast, no age-related difference was found in either the relengthening parameters or the Ca(2+) transient, indicating that relaxation is not directly altered by aging. This latter finding is at variance with previous studies in rat strains with higher rates of pathology. We conclude, therefore, that the primary effect of aging in isolated cardiac myocytes from the F344xBN rat model is a shift in the myosin heavy chain isoform. Changes in relaxation seen in other rat strains may result from compensatory mechanisms induced by pathological conditions.

Parvalbumin gene transfer corrects diastolic dysfunction in diseased cardiac myocytes.

Heart failure frequently involves diastolic dysfunction that is characterized by a prolonged relaxation. This prolonged relaxation is typically the result of a decreased rate of intracellular Ca(2+) sequestration. No effective treatment for this decreased Ca(2+) sequestration rate currently exists. As an approach to possibly correct diastolic dysfunction, we hypothesized that expression of the Ca(2+) binding protein parvalbumin in cardiac myocytes would lead to increased rates of Ca(2+) sequestration and mechanical relaxation. Parvalbumin, which is normally absent in cardiac tissue, is known to act as a soluble relaxing factor in fast skeletal muscle fibers by acting as a delayed Ca(2+) sink. As a test of the hypothesis, gene transfer was used to express parvalbumin in isolated adult cardiac myocytes. We report here that expression of parvalbumin dramatically increases the rate of Ca(2+) sequestration and the relaxation rate in normal cardiac myocytes. Importantly, parvalbumin fully restored the relaxation rate in diseased cardiac myocytes isolated from an animal model of human diastolic dysfunction. These findings indicate that parvalbumin gene transfer offers unique potential as a possible direct treatment for diastolic dysfunction in failing hearts.

Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes.

Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca2+ sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the alpha-MHC isoform to exclusively the beta-MHC isoform. We found a significant desensitization in the Ca2+ sensitivity of tension development in beta-MHC-expressing ventricular myocytes (pCa50=5. 51+/-0.03, where pCa is -log[Ca2+], and pCa50 is pCa at which tension is one-half maximal) compared with that in predominantly alpha-MHC-expressing myocytes (pCa50=5.68+/-0.05). No differences between the 2 groups were observed in the steepness of the tension-pCa relationship or in the maximum isometric force generated. Instantaneous stiffness measurements were made that provide a relative measure of changes in the numbers of myosin crossbridges attached to actin during Ca2+ activation. Results showed that the relative stiffness-pCa relationship was shifted to the right in beta-MHC-expressing myocytes compared with the alpha-MHC-expressing cardiac myocytes (pCa50=5.47+/-0.05 versus 5.76+/-0.05, respectively). We conclude that MHC isoform switching in adult cardiac myocytes alters the Ca2+ sensitivity of stiffness and tension development. These results suggest that the activation properties of the thin filament are in part MHC isoform dependent in cardiac muscle, indicating an additional role for MHC isoforms in defining cardiac contractile function.

Role of Ca2+ and cross-bridges in skeletal muscle thin filament activation probed with Ca2+ sensitizers.

Thin filament regulation of contraction is thought to involve the binding of two activating ligands: Ca2+ and strongly bound cross-bridges. The specific cross-bridge states required to promote thin filament activation have not been identified. This study examines the relationship between cross-bridge cycling and thin filament activation by comparing the results of kinetic experiments using the Ca2+ sensitizers caffeine and bepridil. In single skinned rat soleus fibers, 30 mM caffeine produced a leftward shift in the tension-pCa relation from 6.03 +/- 0.03 to 6.51 +/- 0.03 pCa units and lowered the maximum tension to 0.60 +/- 0.01 of the control tension. In addition, the rate of tension redevelopment (ktr) was decreased from 3.51 +/- 0.12 s-1 to 2.70 +/- 0.19 s-1, and Vmax decreased from 1.24 +/- 0.07 to 0.64 +/- 0.02 M.L./s. Bepridil produced a similar shift in the tension-pCa curves but had no effect on the kinetics. Thus bepridil increases the Ca2+ sensitivity through direct effects on TnC, whereas caffeine has significant effects on the cross-bridge interaction. Interestingly, caffeine also produced a significant increase in stiffness under relaxing conditions (pCa 9.0), indicating that caffeine induces some strongly bound cross-bridges, even in the absence of Ca2+. The results are interpreted in terms of a model integrating cross-bridge cycling with a three-state thin-filament activation model. Significantly, strongly bound, non-tension-producing cross-bridges were essential to modeling of complete activation of the thin filament.

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP.

The chemomechanical coupling mechanism in striated muscle contraction was examined by changing the nucleotide substrate from ATP to CTP. Maximum shortening velocity [extrapolation to zero force from force-velocity relation (Vmax) and slope of slack test plots (V0)], maximum isometric force (Po), power, and the curvature of the force-velocity curve [a/Po (dimensionless parameter inversely related to the curvature)] were determined during maximum Ca2+-activated isotonic contractions of fibers from fast rabbit psoas and slow rat soleus muscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTP as the nucleotide substrate. In addition to a decrease in the maximum Ca2+-activated force in both fiber types, a change from 4 mM ATP to 10 mM CTP resulted in a decrease in Vmax in psoas fibers from 3.26 to 1.87 muscle length/s. In soleus fibers, Vmax was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by the change in nucleotide as the result of a three- to fourfold decrease in the curvature of the force-velocity relationship. The results are interpreted in terms of the Huxley model of muscle contraction as an increase in f1 and g1 coupled to a decrease in g2 (where f1 is the rate of cross-bridge attachment and g1 and g2 are rates of detachment) when CTP replaces ATP. This adequately accounts for the observed changes in Po, a/Po, and Vmax. However, the two-state Huxley model does not explicitly reveal the cross-bridge transitions that determine curvature of the force-velocity relationship. We hypothesize that a nucleotide-sensitive transition among strong-binding cross-bridge states following Pi release, but before the release of the nucleotide diphosphate, underlies the alterations in a/Po reported here.

Determinants of relaxation rate in skinned frog skeletal muscle fibers.

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10 degreesC. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+ concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

Role of calcium and crossbridges in modulation of rates of force development and relaxation in skinned muscle fibers.

The influence of Ca2+ and force generating crossbridges on the kinetics of force development and relaxation was examined in skinned muscle fibers activated by photolytic release of Ca2+ from a caged calcium or inactivated by photolytic uptake of Ca2+ by a caged Ca2+ chelator. In frog fibers at 10 degrees C, decreasing the Ca2+ released from caged calcium to an extent that resulted in 50% of maximum force development produced an approximate seven-fold decrease in the rate of contraction. In contrast decreasing the number of force generating crossbridges by partial extraction of troponin C (TnC) or addition of vanadate caused only minor changes in contraction rate. Thus the rate of force development decreases dramatically with decreases in the Ca2+ level which suggests that a step in the crossbridge cycle may be Ca2+ dependent. The kinetics of relaxation induced by photolysis of diazo-2 was: a) slowed by stabilization of the sarcomeres by repeated releases and re-stretches during contraction and b) accelerated when the amplitude of force development was decreased by decreasing the [Ca2+] which induced a steady contraction. The half time of relaxation decreased by approximately two- to three-fold, when 50% of maximum force was developed. One interpretation of these results is that decreasing the number of force generating crossbridges may speed relaxation by inducing a decreased affinity of TnC for Ca2+ and thus accelerating the Ca2+ dissociation rate from TnC and thereby increasing relaxation rate.

Role of calcium and cross bridges in determining rate of force development in frog muscle fibers.

The influence of Ca2+ and force-generating cross bridges on the kinetics of force development was examined in skinned frog muscle fibers activated by photolytic release of Ca2+ from caged Ca2+ at 10 degrees C. Isometric force development was fit by a double exponential equation with rates of 44.4 s-1 (kc1) and 6.1 s-1 (kc2); kc1 was not significantly different from the rate of force development observed in intact fibers. Maximum activation by caged Ca2+ from preexisting submaximal force produced rates of contraction similar to those observed with maximum activation from zero force. Decreasing the Ca2+ level to an extent that resulted in 50% of maximum force development produced an approximately sevenfold decrease in kc1 and no change in kc2. Partial extraction of troponin C reduced kc1 only slightly (by 16%), whereas decreasing the number of force-generating cross bridges by vanadate did not decrease kc1. Neither treatment altered kc2. Thus the rate of force development increases dramatically with increases in Ca2+ level.

Nucleotide-dependent contractile properties of Ca(2+)-activated fast and slow skeletal muscle fibers.

The relation between single skinned skeletal fiber contractile mechanics and the myosin mechanoenzyme was examined by perturbing the actomyosin interaction with the ATP analog CTP in fibers from both rabbit psoas and rat soleus. Tension, instantaneous stiffness, and the rate of tension redevelopment (ktr), under software-based sarcomere length control, were examined at 15 degrees C for a range of Ca2+ concentrations in both fiber types. CTP produced 94% of the maximum ATP-generated tension in psoas fibers and 77% in soleus fibers. In psoas, CTP also increased stiffness to 106% of the ATP stiffness, whereas in soleus stiffness decreased to 92%. Thus, part of the greater difference between maximum ATP- and CTP-generated tension in soleus fibers appears to be due to a decrease in strongly bound cross-bridge number. Interestingly, although the nucleotide exchange produced substantial increases in the steepness (nH) of the tension- and stiffness-pCa relationships in soleus fibers, only minor changes were seen in psoas fibers. At maximum Ca2+ and nominal P(i) levels, ktr in psoas fibers increased from 11.7 s-1 with ATP to 16.6 s-1 with CTP and in soleus fibers from 4.9 to 8.4 s-1. Increased P(i) levels decreased the maximum Ca(2+)-activated tension in both fiber types and increased the ktr of psoas fibers, but the ktr of soleus fibers was not significantly altered. Thus, although the nucleotide exchange generally produced similar changes in the mechanics, there were significant muscle lineage differences in the tension- and stiffness-pCa relations and in the effects of P(i) on ktr, such that differences in contractile mechanics were lessened in the presence of CTP.

Growth and muscle defects in mice lacking adult myosin heavy chain genes.

The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.