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Rationale: High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce., Objective: To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins., Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values., Conclusions: High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM.

Rationale: The Ca(2+) sensitivity of the myofilaments is increased in hypertrophic cardiomyopathy and other heart diseases and may contribute to a higher risk for sudden cardiac death. Ca(2+) sensitization increases susceptibility to reentrant ventricular tachycardia in animal models, but the underlying mechanism is unknown., Objective: To investigate how myofilament Ca(2+) sensitization creates reentrant arrhythmia susceptibility., Methods and Results: Using hypertrophic cardiomyopathy mouse models (troponinT-I79N) and a Ca(2+) sensitizing drug (EMD57033), here we identify focal energy deprivation as a direct consequence of myofilament Ca(2+) sensitization. To detect ATP depletion and thus energy deprivation, we measured accumulation of dephosphorylated Connexin 43 (Cx43) isoform P0 and AMP kinase activation by Western blotting and immunostaining. No differences were detected between groups at baseline, but regional accumulation of Connexin 43 isoform P0 occurred within minutes in all Ca(2+)-sensitized hearts, in vivo after isoproterenol challenge and in isolated hearts after rapid pacing. Lucifer yellow dye spread demonstrated reduced gap junctional coupling in areas with Connexin 43 isoform P0 accumulation. Optical mapping revealed that selectively the transverse conduction velocity was slowed and anisotropy increased. Myofilament Ca(2+) desensitization with blebbistatin prevented focal energy deprivation, transverse conduction velocity slowing, and the reentrant ventricular arrhythmias., Conclusions: Myofilament Ca(2+) sensitization rapidly leads to focal energy deprivation and reduced intercellular coupling during conditions that raise arrhythmia susceptibility. This is a novel proarrhythmic mechanism that can increase arrhythmia susceptibility in structurally normal hearts within minutes and may, therefore, contribute to sudden cardiac death in diseases with increased myofilament Ca(2+) sensitivity.

Rationale: In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery., Objective: To determine the effects of HNO modification in cardiac myofilament proteins., Methods and Results: The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively., Conclusions: HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

Rationale: At birth, there is a switch from placental to pulmonary circulation and the heart commences its aerobic metabolism. In cardiac myocytes, this transition is marked by increased mitochondrial biogenesis and remodeling of the intracellular architecture. The mechanisms governing the formation of new mitochondria and their expansion within myocytes remain largely unknown. Mitofusins (Mfn-1 and Mfn-2) are known regulators of mitochondrial networks, but their role during perinatal maturation of the heart has yet to be examined., Objective: The objective of this study was to determine the significance of mitofusins during early postnatal cardiac development., Methods and Results: We genetically inactivated Mfn-1 and Mfn-2 in midgestational and postnatal cardiac myocytes using a loxP/Myh6-cre approach. At birth, cardiac morphology and function of double-knockout (DKO) mice are normal. At that time, DKO mitochondria increase in numbers, appear to be spherical and heterogeneous in size, but exhibit normal electron density. By postnatal day 7, the mitochondrial numbers in DKO myocytes remain abnormally expanded and many lose matrix components and membrane organization. At this time point, DKO mice have developed cardiomyopathy. This leads to a rapid decline in survival and all DKO mice die before 16 days of age. Gene expression analysis of DKO hearts shows that mitochondria biogenesis genes are downregulated, the mitochondrial DNA is reduced, and mitochondrially encoded transcripts and proteins are also reduced. Furthermore, mitochondrial turnover pathways are dysregulated., Conclusions: Our findings establish that Mfn-1 and Mfn-2 are essential in mediating mitochondrial remodeling during postnatal cardiac development, a time of dramatic transitions in the bioenergetics and growth of the heart.

Rationale: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied., Objective: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish., Methods and Results: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis., Conclusions: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Rationale: Chronic atrial fibrillation (cAF) is associated with atrial contractile dysfunction. Sarcomere remodeling may contribute to this contractile disorder., Objective: Here, we use single atrial myofibrils and fast solution switching techniques to directly investigate the impact of cAF on myofilament mechanical function eliminating changes induced by the arrhythmia in atrial myocytes membranes and extracellular components. Remodeling of sarcomere proteins potentially related to the observed mechanical changes is also investigated., Methods and Results: Myofibrils were isolated from atrial samples of 15 patients in sinus rhythm and 16 patients with cAF. Active tension changes following fast increase and decrease in [Ca(2+)] and the sarcomere length-passive tension relation were determined in the 2 groups of myofibrils. Compared to sinus rhythm myofibrils, cAF myofibrils showed (1) a reduction in maximum tension and in the rates of tension activation and relaxation; (2) an increase in myofilament Ca(2+) sensitivity; (3) a reduction in myofibril passive tension. The slow beta-myosin heavy chain isoform and the more compliant titin isoform N2BA were up regulated in cAF myofibrils. Phosphorylation of multiple myofilament proteins was increased in cAF as compared to sinus rhythm atrial myocardium., Conclusions: Alterations in active and passive tension generation at the sarcomere level, explained by translational and post-translational changes of multiple myofilament proteins, are part of the contractile dysfunction of human cAF and may contribute to the self-perpetuation of the arrhythmia and the development of atrial dilatation.

Tropomodulin (Tmod)1 caps the pointed ends of actin filaments in sarcomeres of striated muscle myofibrils and in the erythrocyte membrane skeleton. Targeted deletion of mouse Tmod1 leads to defects in cardiac development, fragility of primitive erythroid cells, and an absence of yolk sac vasculogenesis, followed by embryonic lethality at embryonic day 9.5. The Tmod1-null embryonic hearts do not undergo looping morphogenesis and the cardiomyocytes fail to assemble striated myofibrils with regulated F-actin lengths. To test whether embryonic lethality of Tmod1 nulls results from defects in cardiac myofibrillogenesis and development or from erythroid cell fragility and subsequent defects in yolk sac vasculogenesis, we expressed Tmod1 specifically in the myocardium of the Tmod1-null mice under the control of the alpha-myosin heavy chain promoter Tg(alphaMHC-Tmod1). In contrast to Tmod1-null embryos, which fail to undergo cardiac looping and have defective yolk sac vasculogenesis, both cardiac and yolk sac morphology of Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos are normal at embryonic day 9.5. Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos develop into viable and fertile mice, indicating that expression of Tmod1 in the heart is sufficient to rescue the Tmod1-null embryonic defects. Thus, although loss of Tmod1 results in myriad defects and embryonic lethality, the Tmod1(-/-) primary defect is in the myocardium. Moreover, Tmod1 is not required in erythrocytes for viability, nor do the Tmod1(-/-) fragile primitive erythroid cells affect cardiac development, yolk sac vasculogenesis, or viability in the mouse.

Morphogenesis is often considered a function of transcriptional synchrony and the spatial limits of diffusing mitogens; however, physical constrainment by the cell microenvironment represents an additional mechanism for regulating self-assembly of subcellular structures. We asked whether myocyte shape is a distinct signal that potentiates the organization of myofibrillar arrays in cardiac muscle myocytes. We engineered the shape of neonatal rat ventricular myocytes by culturing them on microfabricated fibronectin islands, where they spread and assumed the shape of the island. Myofibrillogenesis followed, both spatially and temporally, the assembly of unique actin networks whose architecture was predictable given the shape of the island. Subsequently, the z lines of the sarcomeres aligned and registered in distinct patterns in different regions of the myocytes in such a way that orthogonal axes of contraction could be distinctly engineered. These data suggest that physical constrainment of muscle cells by extracellular matrix may be an important regulator of myofibrillar organization.

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI(1-192)) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 mum, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic beta-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI(1-192) in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca(2+) sensitivity of the contractile apparatus was increased in preparations containing cTnI(1-192) (pCa(50): 5.73+/-0.03 versus 5.52+/-0.03 for cTnI(1-192) and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI(1-192)-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.

Myosin binding protein-C (MyBP-C) is a poorly understood component of the thick filament in striated muscle sarcomeres. Its C terminus binds tightly to myosin, whereas the N terminus contains binding sites for myosin S2 and possibly for the thin filament. To study the role of the N-terminal domains of cardiac MyBP-C (cMyBP-C), we added human N-terminal peptide fragments to human and rodent skinned ventricular myocytes. At concentrations >10 micromol/L, the N-terminal C0C2 peptide activated force production in the absence of calcium (pCa 9). Force at the optimal concentration (80 micromol/L) of C0C2 was approximately 60% of that in maximal Ca2+ (pCa 4.5), but the rate constant of tension redevelopment (ktr) matched or exceeded (by up to 80%) that produced by Ca2+ alone. Experiments using different N-terminal peptides suggested that this activating effect of C0C2 resulted from binding by the pro/ala-rich C0-C1 linker region, rather than the terminal C0 domain. At a lower concentration (1 micromol/L), exogenous C0C2 strongly sensitized cardiac myofibrils to Ca2+ at a sarcomere length (SL) of 1.9 microm but had no significant effect at SL 2.3 microm. This differential effect caused the normal SL dependence of myofibrillar Ca2+ sensitivity to be reduced by 80% (mouse myocytes) or abolished (human myocytes) in 1 micromol/L C0C2. These results suggest that cMyBP-C provides a regulatory pathway by which the thick filament can influence the activation of the thin filament, separately from its regulation by Ca2+. Furthermore, the N-terminal region of cMyBP-C can influence the SL-tension (Frank-Starling) relationship in cardiac muscle.

In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (approximately 3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from approximately 30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by approximately 10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.

Before birth, the compliance of the heart is limited predominantly by extracardiac constraint. Reduction of this constraint at birth requires that myocardial compliance be determined mainly by the heart's own constituents. Because titin is a principal contributor to ventricular passive tension (PT), we studied the expression and mechanics of cardiac-titin isoforms during perinatal rat heart development. Gel electrophoresis and immunoblotting revealed a single, 3.7-MDa, N2BA isoform present 6 days before birth and an additional, also previously unknown, N2BA isoform of 3.5 to 3.6 MDa expressed in the near-term fetus. These large isoforms rapidly disappear after birth and are replaced by a small N2B isoform (3.0 MDa) predominating in 1-week-old and adult rats. In addition, neonatal pig hearts showed large N2BA-titin isoforms distinct from those present in the adult porcine myocardium. By quantitative reverse transcriptase-polymerase chain reaction, developmentally expressed titin-mRNA species were detected in rat heart. Titin-based PT was much lower (approximately 15 times) in fetal than adult rat cardiomyocytes, and measured PT levels were readily predictable with a model of worm-like chain titin elasticity. Immunofluorescence microscopy showed the extensibility of the differentially spliced molecular spring regions of fetal/neonatal titin isoforms in isolated rat cardiomyofibrils. Whereas the titin-isoform shift by 700 kDa ensures high passive stiffness of the postnatal cardiac myofibrils, the expression of specific fetal/neonatal cardiac-titin isoforms may also have important functions for contractile properties, myofibril assembly or turnover, and myocardial signaling during perinatal heart development.

beta-Adrenergic stimulation increases stroke volume in mammalian hearts as a result of protein kinase A (PKA)-induced phosphorylation of several myocyte proteins. This study investigated whether PKA-induced phosphorylation of myofibrillar proteins directly affects myocyte contractility. To test this possibility, we compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKA. Consistent with previous studies, PKA increased phosphorylation levels of myosin binding protein C and troponin I, and reduced Ca(2+) sensitivity of force. PKA also significantly increased both maximal force (25.4+/-8.3 versus 31.6+/-11.3 microN [P<0.001, n=12]) and peak absolute power output (2.48+/-1.33 versus 3.38+/-1.52 microW/mg [P<0.05, n=5]) during maximal Ca(2+) activations. Furthermore, PKA elevated power output at nearly all loads even after normalizing for the increase in force. After PKA treatment, peak normalized power output increased approximately 20% during maximal Ca(2+) activations (n=5) and approximately 33% during half-maximal Ca(2+) activations (n=9). These results indicate that PKA-induced phosphorylation of myofibrillar proteins increases the power output-generating capacity of skinned cardiac myocytes, in part, by speeding the step(s) in the crossbridge cycle that limit loaded shortening rates, and these changes likely contribute to greater contractility in hearts after beta-adrenergic stimulation.

Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.

We have examined how different degrees (0.5%, 1.0%, 2.5%, 5.0%, and 10.0%) and directions of stretch regulate the turnover and accumulation of contractile proteins in cultured neonatal cardiac myocytes (NCMs). In pulse-chase experiments, stellate-shaped NCMs with random arrays of myofibrils (MFs) exhibited a threshold response to stretch. With respect to unstretched controls, the turnover of the contractile protein pool was suppressed 50% to 100% in stellate NCMs stretched 1.0% to 5.0% and was unaltered in stellate NCMs stretched 0.5% or 10.0%. The posttranslational metabolism of myosin heavy chain (MHC) and actin was regulated in parallel with the total contractile protein pool. The turnover of the cytoplasmic protein pool remained unchanged in response to stretch. NCMs plated onto an aligned matrix of type I collagen expressed an elongated, rod-like cell shape. The MFs of these cells were distributed in parallel with one another along a single unique axis. The tissue-like pattern of organization of these cultures made it possible to assay how specific directions of stretch affected cardiac protein turnover and MF organization. In pulse-chase experiments, stretch in parallel with the MFs did not alter the turnover of the total contractile protein pool, the cytoplasmic protein pool, MHC, or actin. The total cellular concentration of MHC and actin remained constant, and MF alignment was not overtly affected. In contrast, even modest degrees of stretch across the short axis of the MFs suppressed total contractile protein turnover, the turnover of MHC and actin, and promoted the accumulation of these MF subunits. The parallel alignment of MFs deteriorated in myocytes stretched greater than 5%. The characteristic response of aligned myocytes to stretch was not affected by the contractile state of the cells. Isoproterenol (ISO) treatment in concert with stretch in parallel with the MFs modestly accelerated contractile protein turnover. Conversely, contractile protein turnover was suppressed in cells treated with ISO and stretched across the short axis of the MFs. Contractile arrest with nifedipine (NIFED) accelerated total myofibrillar protein turnover. Stretch across the short axis, but not in parallel with the MFs, suppressed protein turnover in cells treated with NIFED. The turnover of the cytosolic proteins remained constant under all conditions assayed. These data suggest that specific directions of stretch may play a crucial role in regulating MF organization and the metabolism of contractile proteins in the cardiac myocyte.

We have tested the hypothesis that decreased functioning of creatine kinase (CK) at sites of energy production and utilization may contribute to alterations in energy fluxes and calcium homeostasis in congestive heart failure (CHF). Heart failure was induced by aortic banding in 3-week-old rats. Myofilaments, sarcoplasmic reticulum (SR), mitochondrial functions, and CK compartmentation were studied in situ using selective membrane permeabilization of left ventricular fibers with detergents (saponin for mitochondria and SR and Triton X-100 for myofibrils). Seven months after surgery, animals were in CHF. A decrease in total CK activity could be accounted for by a 4-fold decrease in activity and content (Western blots) of mitochondrial CK and a 30% decrease in M isoform of CK (MM-CK) activity. In myofibrils, maximal force, crossbridge kinetics, and alpha-myosin heavy-chain expression decreased, whereas calcium sensitivity of tension development remained unaltered. Myofibrillar CK efficacy was unchanged. Calcium uptake capacities of SR were estimated from the surface of caffeine-induced tension transient (SCa) after loading with different substrates. In CHF, SCa decreased by 23%, and phosphocreatine was 2 times less efficient in enhancing calcium uptake. Oxidative capacities of the failing myocardium measured as oxygen consumption per gram of fiber dry weight decreased by 28%. Moreover, the control of respiration by creatine, ADP, and AMP was severely impaired. Our observations provide evidence that alterations in CK compartmentation may contribute to alterations of energy fluxes and calcium homeostasis in CHF.

The organization of actin into striated fibers (myofibrils) is one of the major features of cardiac hypertrophy. However, its signal transduction mechanism is not well understood. Although Rho-family small G proteins have been implicated in actin organization in many cell types, it is not fully elucidated whether Rho mediates the organization of actin fibers by hypertrophic stimuli in cardiac myocytes. Therefore, we examined (1) whether Rho is activated by the hypertrophic stimulus, angiotensin II (Ang II), and (2) whether Rho mediates the Ang II-induced organization of actin fibers in cultured neonatal rat cardiac myocytes. Treatment of myocytes with Ang II caused a rapid formation of both striated (mature myofibrils) and nonstriated (premyofibrils) actin fibers within 30 minutes, as determined by phalloidin stainings of the polymerized actin and troponin T stainings. Immunoblot analyses and immunostainings have indicated that cardiac myocytes express RhoA, but RhoB is undetectable. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II, consistent with activation of RhoA by Ang II. Incubation of myocytes with exoenzyme C3 for 48 hours completely ADP-ribosylated Rho in vivo. The C3 treatment abolished formation of premyofibrils induced by Ang II, suggesting that Ang II causes premyofibril formation via a Rho-dependent mechanism. The Ang II-induced mature myofibril formation was only partly abolished by C3. Expression of constitutively active RhoA (V14RhoA) caused the formation of premyofibrils but not mature myofibrils. The C3 treatment inhibited Ang II-induced atrial natriuretic factor induction, whereas it had no effect on c-fos induction. These results indicate that RhoA is activated by Ang II and mediates the Ang II-induced formation of premyofibrils and induction of a subset of genes. Distinct signaling mechanisms seem to be responsible for striated mature myofibril formation by Ang II.

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.

Tropomodulin is a 40.6-kD protein that colocalizes with actin filament pointed ends in skeletal muscle. We report the sequence of two partial-length complementary DNA (cDNA) clones of rat cardiac tropomodulin that cover 90% of the coding region. The cDNA sequence is 90% conserved between human and rat, with the predicted amino acid sequence similarity even higher at 95%. Anti-tropomodulin antibodies label a single polypeptide with an apparent mobility of 43,000 in Western blot analysis of rat cardiac muscle. Immunofluorescence experiments using this anti-tropomodulin antibody result in labeling that is coincident with thin filament ends, as demonstrated by double localization with alpha-actinin antibody. Tropomodulin protein is organized into a sarcomeric staining pattern with the earliest appearance of myofibrils in rat cardiocytes. The localization of tropomodulin protein at or near thin filament ends led us to examine the distribution of tropomodulin messenger RNA (mRNA) during myofibrillar development in vitro. Fluorescent in situ hybridization experiments using tropomodulin cDNA probe in cardiocytes that have been cultured for 3 to 5 days show a distribution of large mRNA patches. The cytoplasmic location of tropomodulin mRNA at this time, which bears no relation to the developed myofibrils, suggests that tropomodulin protein is targeted to thin filament ends rather than using localized translational machinery. However, the distribution of tropomodulin mRNA in cultured cardiocytes changes over the next 2 weeks from large perinuclear patches to small concentrations arranged along myofibrils throughout the cell. The reorganization of tropomodulin mRNA throughout the cardiocyte appears to be distinct from the pattern of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same time period. Increasing intracellular density of myofibrils within developing cardiocytes may lead to redistribution of selected mRNAs for localized translation.

Spontaneous oscillations observed in various heart muscle preparations are widely thought to be triggered by spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). Here, we report undamped propagated oscillations that occur in the absence of SR. In single cardiac myofibrils treated with Triton X-100 to remove SR and held isometrically, partial activation initiated periodic fluctuations of sarcomere length persisting up to 1 hour. Oscillation characteristics could be readily quantitated by virtue of the small size of the preparation. In an individual sarcomere, the oscillation cycle generally consisted of a slow shortening phase, followed by a phase of rapid lengthening. Oscillations usually propagated along the myofibril--frequently along the entire specimen--in a wavelike fashion (average velocity, 12.3 microns/s at 10 degrees C; Q10, approximately 1.3). The oscillation period was 2.30 and 1.72 seconds at 10 degrees and 20 degrees C, respectively, and was insensitive to stretch. The average oscillation amplitude, which was temperature independent, decreased with stretch from more than 20% of the mean sarcomere length at lengths below 2 microns to zero beyond a sarcomere length of 3 microns. Stiffening of the Z line by labeling with anti-alpha-actinin resulted in a dose-dependent decrease of oscillation amplitude, while the period was not affected. Tension oscillations could not be detected in single myofibrils but were frequently detectable in myofibril doublets, where the oscillation magnitude (approximately 1 microgram) was above the noise floor. Addition of 10 mumol/L ryanodine to the activating solution did not alter oscillation characteristics, as expected, since the oscillations are unrelated to SR calcium release. On the basis of our results, we consider a mechanism for the oscillations in which a length dependence of myofibrillar Ca2+ sensitivity and a dynamic Z-line structure are essential.

The sarcomeric proteins and organization of cardiac myofibrils appeared intact in multiple unrelated patients with hypertrophic cardiomyopathy. In two subjects demonstrating the missense mutation at position 403 (Arg to Gln) in the beta-myosin heavy chain gene, total myosin and immunoreactive beta-myosin heavy chain levels were similar to those found in other patients with hypertrophic cardiomyopathy and various disease control subjects. No alteration in expression of the cardiac alpha-myosin heavy chain gene was observed. These results are consistent with the examined myosin heavy chain mutation, permitting proper accumulation and assembly of myosin while primarily impairing contractile function. The characteristic myocyte disarray would appear likely to be a secondary consequence of the mutations.

We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.

The effect of temperature on the force-sarcomere velocity relation (20 degrees, 25 degrees, and 30 degrees C) and maximum velocity of sarcomere shortening (Vo; range 15 degrees-35 degrees C) was studied in trabeculae from rat heart. Sarcomere length and Vo were measured by laser diffraction techniques. Sarcomere length and sarcomere velocity, determined from each of the first-order diffraction lines, differed by less than 4%. Slack sarcomere length in the trabeculae appeared to be 1.9 microns. Isovelocity release techniques were used to obtain sarcomere velocity and Vo directly. Sarcomere velocity was measured at SL = 1.9-2.0 microns for elimination of contributions of parallel elastic force and restoring force to the external load of the sarcomeres. Peak twitch force development (Fo) was maximal (Fo-max) at 25 degrees C at [Ca2+]o = 1.5 mM. Lowering of the temperature below 25 degrees C led to development of spontaneous sarcomere activity and depression of Fo; both responses could be prevented by the addition of 0.5 mM procaine. Increase of temperature above 25 degrees C reduced twitch duration and Fo. Hill's rectangular hyperbola fitted the force-velocity data if the load during shortening was less than 70% of Fo. Vo appeared to be independent of the level of activation at all temperatures when Fo was maintained above 90% of Fo-max, either by an increase of [Ca2+]o (to 3.0 mM) or by paired pulse stimulation. Vo increased with increasing temperature; the parameter a, calculated from force-velocity relations measured at 20 degrees, 25 degrees, and 30 degrees C, decreased with increasing temperature. The Arrhenius plot of Vo was studied in detail over a wider temperature range (15 degrees-35 degrees C) and in smaller temperature increments. The relation was linear between 18 degrees and 33 degrees C; the observed Q10, defined as the ratio of Vo measured at temperature (T) over Vo at T-10 degrees C, was 4.6 A Q10 of 4.6 for Vo is consistent with the reported temperature dependence of rat cardiac actin-activated myosin ATPase, which suggests that the same reaction step may limit the activity of the enzyme in vitro and during shortening of the cardiac sarcomeres at zero external load.

Postnatal development of the mammalian heart is associated with changes in the population of isoforms of the thin filament proteins. We correlated the change in thin filament proteins, which occur in rabbit hearts between 5 days and 22 days of age, with changes in Ca2+ dependence of myofibrillar ATPase activity, force generation, and troponin C Ca2+ binding. The preparations derived from the 5-day-old animals exhibited a high molecular weight isoform of troponin T not found in the hearts of the 22-day-old animals. Other troponin T isoforms were also found to be present in different relative amounts. No other major differences in thin filament protein composition could be identified. Compared with the 5-day-old rabbit heart preparations, the ATPase activity of myofibrils from 22-day-old rabbit hearts exhibited a reduced Ca2+ sensitivity. The pCa50 (negative log of the half-maximal-activity free Ca2+) of the MgATPase activity was shifted by 0.15 pCa units with maturation. Maturation of the myofibrils was also associated with an increased effect of Mg2+ on pCa50. On increasing the Mg2+ from 2 to 10 mM at constant MgATP2-, the pCa50 of 5-day myofibrils was increased (shifted to the right) by 0.39 pCa units for 5-day-old rabbit hearts and 0.45 pCa units for 22-day-old rabbit hearts. Although similar changes in pCa50 of force developed by myofibrils were marginally significant, fibers from hearts of 5-day-old rabbits exhibited a greater Hill coefficient than hearts from 22-day-old rabbits (3.0 vs. 2.1). Despite the increased sensitivity of 5-day-old rabbit hearts to Ca2+, these hearts exhibited significantly less Ca2+ bound to myofibrillar troponin C than did the 22-day-old rabbit hearts. Moreover, the models that best described the Ca2+ binding data are different for the two age groups. Our data indicate that the Ca2+ activation and Ca2+ binding properties of myofibrillar troponin C are altered in developing cardiac myofibrils and that the changes in these properties may be influenced by changes in the troponin T isoforms present in the myofibril.

Sarcomere shortening and ultrastructure of intact isolated myocytes from ventricles of three-week-old and adult rabbits were examined. Cells were fixed and embedded, and after measuring their sarcomere shortening in response to electrical stimulation, they were examined in serial thin sections by electron microscopy. This structure-function analysis showed that adult cells were significantly larger, had longer rest sarcomere lengths, greater amount and velocity of sarcomere shortening, greater velocity of reextension, and shorter contraction duration than immature cells. In immature myocytes, a thin outer shell of myofibrils enveloped a central mass of mitochondria and nuclei, but in adult cells, the cytoskeleton divided the cell into compartments with the mitochondria arranged around and interspersed among the myofibrils. The different arrangement of the organelles and the cytoskeleton at the two ages may account for the shorter rest sarcomere length in the young myocytes and may confer differing internal loads that contribute to their smaller amount and velocity of sarcomere shortening. The corbular and longitudinal sarcoplasmic reticulum were less demarcated in immature than in adult cells. Myocytes from both ages showed postextrasystolic potentiation, suggesting that the sarcoplasmic reticulum modulates calcium at both ages. Restitution of contractility between contractions, obtained by measuring sarcomere shortening of interpolated extrasystoles, was faster in immature than in adult cells and may reflect the structural differences in the sarcoplasmic reticulum. The developmental differentiation in the sarcoplasmic reticulum suggests that changes in compartmentalization of calcium and in the distribution of putative calcium-release sites contribute to the increased contractility of adult myocytes.

It generally has been thought that the relatively high resting tension characteristic of cardiac tissue resides in structures (collagen, elastin) external to the individual cardiac cells, but the evidence to support this conclusion has been indirect, since the resting tension of intact single cardiac cells has not been determined previously. The purpose of the present investigation was to determine the resting tension (stress)-sarcomere length relationships of single intact frog atrial cells. For tension determinations, a single cell was attached between two poly-L-lysine coated glass beams; one beam served as a compliant calibrated cantilevered force beam, and length changes were imposed on the cell by movement of the other beam. Coventional bright-field light microscope techniques were used to view the cell, the sarcomere pattern within the cell, and the position of the force beam. The resting tension of the intact cell increased from a value of about 10 nN at a sarcomere length of 2.35 microns to a value of about 130 nN at a sarcomere length of 3.45 microns. Lagrangian and Eulerian resting stress-sarcomere length relationships were computed from the resting tension-sarcomere length relationships. The Lagrangian stress increased from a value of about 0.6 mN/mm2 at a sarcomere length of 2.35 microns to a value of about 7 mN/mm2 at a sarcomere length of 3.45 microns. These values of stress are about 8- to 30-fold less than those previously reported for intact frog atrial tissue and indicate that the resting tension of intact frog atrial preparations resides primarily in structures external to the individual cardiac cell.

Recent experiments using laser diffraction techniques to determine the time course and extent of sarcomere shortening in thin bundles of cardial tissue have given results which suggest that the velocity of sarcomere shortening in cardiac muscle is independent of the developed force (Nassar et al., 1974; Krueger and Pollack, 1975). However, the anatomical complexity of the intact tissue precludes a definite interpretation of the data, since the exact relationship between the force being borne by the total tissue to the force being borne by any observed group of sarcomeres is uncertain. The single frog atrial cell provides a simple cardiac preparation in which the relationship between sarcomere velocity and sarcomere force is well defined, since these cells are only 1-2 myofibrils wide. The purpose of the present investigation was to determine if sarcomere velocity in the single frog atrial cell is dependent on force by measuring the time course of sarcomere shortening in single cells under conditions in which the cell developed markedly different forces. The results presented in this paper give direct evidence that the velocity of sarcomere shortening in the single cardiac cell depends on the force being developed by the sarcomeres. Thus, cardiac sarcomeres have a type of force-velocity relationship, although the exact nature of this relationship could not be determined in these experiments.

A model of the left ventricle which combines a spheroidal geometry with a spatial fiber angle distribution is presented. The mechanics of each muscle fiber is described by its passive stress-strain relationship, active stress-strain relationship, and an activation function (half a sinusoid) which represents the time-dependent degree of activation of the fiber. A stress-strain rate relationship which characterizes the muscle fibers is used to calculate the mechanics of left ventricular contraction during ejection. Furthermore, a radial electrical signal propagation from the endocardium to the epicardium is used here as a first approximation to the actual depolarization sequence. The model is used to describe the process of contraction throughout the systole. The different calculated parameters and indices of left ventricular function are presented and discussed for different preloading, afterloading and contractility conditions. The maximum elastance is found to be an optimal macroscale parameter of contractility, as it is completely preload and afterload independent, and is a good reflection of the active microscale sarcomere stress-strain relationship.

We studied sarcomere performance in single isolated intact cardiac cells using techniques that allow direct measurement of sarcomere length and force. This investigation dealt primarily with sarcomere performance during twitch contractions under lightly loaded conditions. In such contractions, there was a significant portion of the contraction in which sarcomere shortening occurred at constant velocity over a significant range of sarcomere lengths. The constant velocity phase of shortening was followed by a phase of shortening in which sarcomere velocity decreased markedly. Both the velocity and extent of sarcomere shortening depended on the stimulus parameters used to excite the cell. With threshold stimulation, sarcomere velocities during the constant velocity phase of shortening ranged from 1 to 5.5 micron/sec in different cells and significant slowing of sarcomere shortening began at sarcomere lengths of 1.8-2.0 micron. In contrast, when cells were stimulated with a long duration stimulus (200 msec) of large current strength, sarcomere velocities during the constant velocity phase ranged from 6 to 12 micron/sec, and significant slowing did not occur until a sarcomere length of about 1.6 micron was reached. The threshold stimulus strength-stimulus duration relationship was determined on the single cell, and it was found to be of the type expected for a cell having an intact excitable membrane capable of generating an action potential when depolarized to a fixed voltage threshold. The data presented in this paper give direct evidence that the lightly loaded cardiac sarcomere has a velocity of shortening which depends on the level of contractile activation but is independent of sarcomere length at sarcomere lengths greater than about 1.6 micron.

In experiments designed to achieve maximal activation, the active force/cell cross-sectional area in tissues prepared from the swine carotid media was 6.7 +/- 0.3 (sd) X 10(5) N/m5. This value exceeds that reported for other vertebrate muscle cells and is striking because of the low smooth muscle myosin content. The hypothesis that high force generation may, in part, reflect an increase in the crossbridge duty cycle, i.e., the fraction of the cycle during which force is generated, was tested by determining the rate of force redevelopment after a step shortening and the ration of the load-bearing capacity of the contractile system to the developed stress during the course of isometric contractions. Maximal crossbridge cycling rates estimated by the rate of force redevelopment occurred 30 seconds after the onset of a high K+-induced contraction, and decreased thereafter, although the load-bearing capacity or maximum active stress was maintained. These results from isometric experiments support the hypothesis and provide further evidence that attached, non-cycling crossbridges contribute to force maintenance in tonically contracting arterial smooth muscle.

We compared the response of ventricular muscle from adult and neonatal rats to hypercapnic acidosis. In adult muscle, acidosis caused an initial rapid fall of developed tension to 30 +/- 5% of control (mean +/- SEM, n = 6). However, tension recovered slowly to a steady state that was 56 +/- 6% of control. In neonatal muscle, acidosis caused a significantly smaller initial fall in tension to 43 +/- 3% (n = 8, p less than 0.05), but the tension then showed a subsequent slower fall to a steady state that was 29 +/- 4% of control, significantly less than in the adult (p less than 0.01). We have attempted to identify the mechanisms underlying these differences in response. In detergent-skinned myofibrils, reducing the pH from 7.0 to 6.5 caused a reduction in the pCa50 of 0.61 units in the adult muscle, but only 0.27 units in the neonatal ventricular muscle. Myofibrillar Ca2+ sensitivity in neonatal ventricular muscle is thus less susceptible to the effects of acidic pH than that of adult muscle. Since intracellular pH decreases rapidly on application of increased external CO2, these results are consistent with the finding that, initially, developed tension in neonatal muscles is less sensitive to the effects of acidosis. Sodium dodecylsulfate gel electrophoresis of myofibrillar preparations from adult and neonatal rats demonstrated differences in thin filament proteins, including troponin I, which may underlie the observed differences in Ca2+ sensitivity. In adult rat ventricular muscles, the slow recovery of tension during acidosis is associated with an increase in the amplitude of the Ca2+ transients to 263 +/- 34% of control (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

When cardiac muscle shortens during a contraction, the duration of mechanical activity is abbreviated (shortening deactivation), but the duration of the action potential is prolonged. Neither of these phenomena is fully understood, but both may be related to changes in the myoplasmic free calcium concentration. In these experiments, isolated papillary muscles from cats and ferrets were allowed to contract under various mechanical conditions while myoplasmic calcium was monitored with aequorin, or in parallel experiments the membrane potential was recorded with microelectrodes or a sucrose gap. When shortening occurred, myoplasmic calcium was increased and the membrane potential was more positive than in isometric contractions. The changes in calcium apparently precede the depolarization. We propose that muscle shortening reduces calcium binding to the contractile proteins and leads to a rise in myoplasmic calcium, and that this rise in myoplasmic calcium activates an inward current leading to the observed changes in the action potential. These processes may be important contributory factors in some arrhythmias.

To investigate the extent to which the properties of the cardiac myofibrils contribute to the length-force relation of cardiac muscle, we determined the sarcomere length-force relations for rat ventricular trabeculae both before and after the muscles were skinned with the detergent Triton X-100. Sarcomere length was measured continuously by laser diffraction. In the unskinned trabeculae stimulated at 0.2 Hz, the relation between active force and sarcomere length at an extracellular calcium concentration of 1.5 mM was curved away from the sarcomere length axis, with zero force at sarcomere length of 1.5-1.6 micron. At 0.3 mM calcium, the sarcomere length-force relation was curved toward the sarcomere length axis. Chemical skinning of the muscle with 1% Triton X-100 in a "relaxing solution" caused an increase in intensity and decrease in dispersion of the first order diffraction beam, indicating an increased uniformity of sarcomere length in the relaxed muscle. During calcium-regulated contractures in the skinned muscles, the central sarcomeres shortened by up to 20%. As the calcium concentration was increased over the range 1-50 microM, the relation between steady calcium-regulated force and sarcomere length shifted to higher force values and changed in shape in a manner similar to that observed for changes in extracellular calcium concentration before skinning. The sarcomere length-force relations for the intact muscles at an extracellular calcium concentration of 1.5 mM were similar to the curves at calcium concentration of 8.9 microM in the skinned preparations, whereas the curves at an extracellular calcium concentration of 0.3 mM in intact muscles fell between the relations at calcium concentrations of 2.7 and 4.3 microM in the skinned preparations. A factor contributing to the shape of the curves in the skinned muscle at submaximal calcium concentrations was that the calcium sensitivity of force production increased with increasing sarcomere length. The calcium concentration required for 50% activation decreased from 7.71 +/- 0.52 microM to 3.77 +/- 0.33 microM for an increase of sarcomere length from 1.75 to 2.15 micron. The slope of the force-calcium concentration relation increased from 2.82 to 4.54 with sarcomere length between 1.75 and 2.15 micron. This change in calcium sensitivity was seen over the entire range of sarcomere lengths corresponding to the ascending limb of the cardiac length-force relation. It is concluded that the properties of the cardiac contractile machinery (including the length-dependence of calcium sensitivity) can account for much of the shape of the ascending limb in intact cardiac muscle.

The effects of stretches and releases on the contractile performance of isolated single frog atrial cells (Rana catesbeiana) were investigated. A stretch or release was imposed on the cell--either during a contraction (test) or before the onset of contraction (control)--and the contractile performance (length, velocity and force) of the test contraction was compared with that of the control contraction to determine whether the stretch or release imposed on the contracting cell altered the contractility of the cell. We found that the velocity of cell (and sarcomere) shortening for the remainder of the test contraction following either a stretch or release was markedly less than that occurring at the same time in the control contraction. This decreased velocity occurred even though the force in the test contraction was less than that in the control contraction and the sarcomere length was longer in the test contraction than in the control contraction. These results indicate that after a stretch or release imposed on the contracting cell, the force-velocity relationship at any given length and time is depressed than had the stretch or release not been imposed on the contracting cell. Thus, stretches and releases applied to the contracting single cardiac cell either produce a long-term depression in the contractility of the cell, or that the contractility at any given time and sarcomere length depends markedly on the history of the contraction.

We studied sarcomere performance in single isolated intact frog atrial cells using techniques that allow direct measurement of sarcomere length and force. The purpose of this investigation was to determine whether length-dependent alterations in contractile activation occur in the single isolated cardiac cell. This was accomplished by determining the effect of initial sarcomere length on the time course of sarcomere shortening and force development during auxotonic twitch contractions. The results presented in this paper demonstrate that the velocity of sarcomere shortening, the rate of force development, and the magnitude of force development during auxotonic twitch contractions all increase as initial sarcomere length increases over the range of about 2 micrometers to greater than 3 micrometers. These results indicate that the level of contractile activation increases as initial sarcomere length increases. Also, results are presented that indicate that the rate of increase of contractile activation during a twitch contraction also increases as initial sarcomere length increases. These length-dependent effects on contractile activation in conjunction with the slow time course of contractile activation cause the force-velocity-length relationship to be time-dependent: i.e., the velocity of sarcomere shortening at a given sarcomere length and load depends on the time during the contraction when the sarcomere reaches that length. The results suggest that length-dependent alterations in contractile activation may play a major role in the improved contractile performance that accompanies an increase in initial sarcomere length in cardiac muscle.

Traditionally it is assumed that during cardiac depolarization the macroscopic current generators that produce electrocardiographic voltages can be represented as a uniform double-layer source, coincident with the macroscopic boundary between resting and depolarized cardiac fibers as measured with extracellular electrodes ("uniform" hypothesis). A segment of this boundary is thus considered as a current dipole oriented perpendicular to the boundary. We present evidence that, contrary to the above, the effective dipoles largely parallel the long axes of cardiac fibers ("axial" hypothesis). Calculated potentials in volume conductors differ markedly in the two cases. The magnitudes of rapid local "intrinsic" deflections also differ markedly. In our experiments, potential fields prodlced by stimulation at several cardiac sites and measured magnitudes of intrinsic deflections during normal depolarization and that caused by stimulation support the axial hypothesis and are incompatible with the uniform hypothesis. Our results suggest that axial orientation of sources is sufficiently strong so that predictions assuming the uniform hypothesis would be seriously in error, although the axial theory alone does not exactly describe all the measured potentials. Axial orientation of current generators must be considered in quantitative prediction of electrocardiographic potentials. tfurther study of the geometry of the intracellular depolarization boundary and its relation to fiber direction and to the frequency of lateral intercellular junctions is required to describe the generators exactly.

Kinetics of sarcomere movement were studied in real-time by laser diffraction. Instantaneous sarcomere shortening was measured during afterloaded twitches simultaneously with instantaneous shortening and tension of the whole trabecula excised from rat right ventricle. Resting sarcomere length at optimal length was 2.20 +/- 0.02 micron (mean +/- SEM). Maximum amplitude of sarcomere shortening was 0.30 +/- 0.01 and 0.16 +/- 0.01 micron, respectively, in twitches loaded with preload only, and in "isometric" twitches. When the isotonic load (expressed as a percentage of maximum isometric force TF) increased, the maximum velocity of sarcomere relaxation max Vr (micron/sec) decreased: max Vr = -4 exp (-2.5 X 10(-2) % TF); r = 0.95. The time course of sarcomere relaxation appeared to be progressively delayed when the total load increased from preload only up to "isometric" load. Sarcomere relaxation occurred in two successive exponential phases, a rapid phase [time constant (msec): tau 1] followed by a slower one (time constant: tau 2). When the total load increased, tau 1 increased and tau 2 decreased according to the linear relations: % TF = 0.2 tau 1 + 4.8 (r = 0.83) and % TF = -0.1 tau 2 + 157 (r = 0.95). The relative predominance of both the time course and the amplitude of these two phases depended upon the level of total load. The rapid process predominated at low load, the slow one at high load. The role of load and/or shortening in the time course of these two phases is discussed.

A series of experiments was carried out to determine the effects of load variation on the character of stepwise shortening. We imposed afterloaded isotonic contractions on rat ventricular trabeculae, and measured the effect of load on pause duration, i.e., on the duration of the periods during which there was no sarcomere shortening. Sarcomere lengths were measured by optical diffraction. Increases of load brought about increases of pause duration; the relation was linear. The relation did not appear to depend on time during contraction, but did depend on sarcomere length: for a given load, pauses were longer at shorter sarcomere lengths. In a supplementary protocol in which we measured the dynamics of the central segment of the muscle during muscle isometric contraction, we found that the velocity of sarcomere shortening during the shortening step was approximately independent of load. These results provide a framework for interpretation of muscle force-velocity relations: diminished velocity at high load may be the result of increased pause durations.

The aim of experiments described here was to test whether deactivation of cardiac myofibrils in acidic pH is associated with decreases in amounts of calcium bound to myofilament troponin. We determined the amounts of myofibrillar bound calcium attributable to troponin, from measurements of calcium binding to myofibrils and to myosin and from determination of the troponin C content of the myofibrillar preparations (0.40 nmol troponin C/mg protein). In measurements done at 2 mM free magnesium, 2 mM (magnesium-adenosine triphosphate, ionic strength 0.12, 22 degrees C, the pCa50 (-log of the half maximally activating molar free calcium) for myofibrillar magnesium-adenosine triphosphatase activity was 5.87 at pH 7.0, 5.49 at pH 6.5, and 5.04 at pH 6.2. This change in calcium sensitivity of myofibrillar magnesium-adenosine triphosphatase activity was present whether or not ethyleneglycol-bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, was used to buffer the free calcium and whether or not myofibrillar troponin I had been phosphorylated by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. However, the change in pCa50 of myofibrillar adenosine triphosphatase activity induced by acidic pH, was greater when free magnesium was reduced from 2.0 to 0.05 mM, and less when free magnesium was increased from 2.0 mM to 10 and 15 mM. The change in pCa50 with acidic pH was less if the ionic strength was reduced from 0.12 to 0.035 M. The magnesium-adenosine triphosphatase activity of troponin/tropomyosin-free myofibrils was independent of pCa and unaffected by a reduction of pH from 7.0 to 6.5. The affinity of myofibrillar troponin C for calcium decreased as pH was reduced from 7.0 to 6.5 and to 6.2 with and without ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and in a manner predicted from the effect of acidic pH on pCa50 for myofibrillar activation. Our results are consistent with the idea that at least part of the mechanism responsible for deactivation of the adenosine triphosphatase activity of cardiac myofilaments in acidic pH is a reduction in the affinity of myofibrillar troponin C for calcium.