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Gene editing is moving rapidly from a highly useful laboratory-based tool toward human clinical application. In vivo gene editing has been documented in mouse models of Duchenne muscular dystrophy (DMD), where skeletal and cardiac muscle editing produces internally truncated dystrophin. A recent report now demonstrates that editing the dystrophin gene in the heart improves cardiac function, paving the path to in vivo application of cardiac gene correction. Article, see p 923 Mutations in the gene encoding dystrophin cause DMD and its associated cardiomyopathy. Large deletions, which span multiple exons, are the most common cause of DMD, and these deletions disrupt dystrophin’s reading frame and ablate its expression. Dystrophin binds actin at its N-terminus and with its C-terminus, dystrophin connects to the plasma membrane of myocytes. The central portion of dystrophin is composed of multiple spectrin repeat domains, and mutations that interrupt the internal spectrin repeat domains and leave intact the N- and C-terminus result in a milder form of disease called Becker muscular dystrophy.1 A major therapeutic approach for DMD aims to restore the reading frame by converting DMD-associated frame-disrupting mutations into those that produce internally deleted but more functional BMD-associated dystrophins. In 2016, the Food and Drug Administration granted approval for eteplirsen, which uses antisense technology to restore dystrophin’s reading frame.2 Antisense treatments act on RNA and, therefore, require regular repeated dosing to induce the expected molecular effect. In contrast, gene editing corrects DNA, and if directed to the correct cell type, it can afford a permanent treatment with a single application. Gene editing using CRISPR/Cas9 induces double-stranded breaks in DNA, which typically rejoin at predictable sites using nonhomologous end joining.3 Alternatively, when CRISPR/Cas9 is activated in the presence of a corrective template, homology-directed repair can generate specific base pair changes, like those required to fully …

Integrins mechanically link the cytoskeleton to the extracellular matrix in cardiac myocytes and are thereby involved in mechanotransduction. Integrins appear to be necessary for cardiac myocyte hypertrophy. To determine the effect of increased integrin ligation and signaling on adult cardiac function, a heart-specific truncated α 5 integrin (gain of function) was conditionally expressed in mice. Four days later, we observed an 80% reduction in amplitude of the QRS complex, profound systolic dysfunction, decreased connexin43, loss of gap junctions, and abnormal intercalated discs. Surprisingly, isolated left ventricular myocytes contracted normally and exhibited normal Ca 2+ transients. This suggested that cell/cell electrical and/or mechanical coupling was disrupted. To distinguish electrical from mechanical coupling deficits, we compared the papillary muscle force generated by electrically stimulated versus rapid cooling contractions in which intracellular Ca 2+ is released without electrical depolarization. Both were decreased in the transgenic muscle. However, electrically stimulated contractions were more significantly reduced than rapid cooling contractures. This suggests a component of cell/cell electrical uncoupling. Optical mapping revealed a loss of the normal elliptical isochronal activation pattern implying a loss of preferential conduction through gap junctions. For the first time, we have shown that integrins can regulate both mechanical and electrical coupling in the adult heart, even in the absence of primary hemodynamic alterations. Furthermore, we demonstrated that unregulated integrin activation leads to both contractile dysfunction and arrhythmias.

Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl¯-HCO3¯ exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness., Facultad de Ciencias Médicas, Centro de Investigaciones Cardiovasculares

Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC–Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca 2+ -dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca 2+ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-ε after PHE or ET exposure of WT preparations. In contrast, myofilament-associated PKC-ε was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilament-associated PKC-β were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca 2+ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-β.

Abstract —The intramural dynamics of ventricular fibrillation (VF) remain poorly understood. Recent investigations have suggested that stable intramural reentry may underlie the mechanisms of VF. We performed optical mapping studies of VF in isolated swine right ventricles (RVs) and left ventricles (LVs). Nine RV walls were cut obliquely in their distal edge exposing the transmural surface. Six LV wedge preparations were also studied. Results showed that intramural reentry was present. In RV, 28 of 44 VF episodes showed reentry; 15% of the activation pathways were reentrant. Except for 4 episodes, reentry was transmural, involving subendocardial structures as the papillary muscle (PM) or trabeculae. In LV, reentry was observed in 27 of 27 VF episodes; 23% of the activations were part of reentrant pathways ( P

Abstract —Electric shock is the only effective therapy against ventricular fibrillation. However, shocks are also known to cause electroporation of cell membranes. We sought to determine the impact of electroporation on ventricular conduction and defibrillation. We optically mapped electrical activity in coronary-perfused rabbit hearts during electric shocks (50 to 500 V). Electroporation was evident from transient depolarization, reduction of action potential amplitude, and upstroke dV/dt. Electroporation was voltage dependent and significantly more pronounced at the endocardium versus the epicardium, with thresholds of 229±81 versus 318±84 V, respectively ( P =0.01, n=10), both being above the defibrillation threshold of 181.3±45.8 V. Epicardial electroporation was localized to a small area near the electrode, whereas endocardial electroporation was observed at the bundles and trabeculas throughout the entire endocardium. Higher-resolution imaging revealed that papillary muscles (n=10) were most affected. Electroporation and conduction block thresholds in papillary muscles were 281±64 V and 380±79 V, respectively. We observed no arrhythmia in association with electroporation. Further, preconditioning with high-energy shocks prevented reinduction of fibrillation by 50-V shocks, which were otherwise proarrhythmic. Endocardial bundles are the most susceptible to electroporation and the resulting conduction impairment. Electroporation is not associated with proarrhythmic effects and is associated with a reduction of vulnerability.

Abstract —Because the amount and structure of type I collagen are thought to affect the mechanics of ventricular myocardium, we investigated myocardial collagen structure and passive mechanical function in the osteogenesis imperfecta murine ( oim ) model of pro-α2(I) collagen deficiency, previously shown to have less collagen and impaired biomechanics in tendon and bone. Compared with wild-type littermates, homozygous oim hearts exhibited 35% lower collagen area fraction ( P P P oim left ventricular (LV) collagen concentration was 45% lower ( P P oim than wild-type ( P =NS). Uniaxial stress-strain relations in resting right ventricular papillary muscles exhibited 60% greater strains ( P P =0.05), and 64% higher nonlinearity ( P oim . Mean opening angle, after relief of residual stresses in resting LV myocardium, was 121±9 degrees in oim compared with 45±4 degrees in wild-type ( P oim was 23±8 degrees greater than wild-type ( P oim is associated with significantly decreased fiber and chamber stiffness despite modestly increased collagen cross-linking. Altered myofiber angles and residual stress may be beneficial adaptations to these mechanical alterations to maintain uniformity of transmural fiber strain. In addition to supporting and organizing myocytes, myocardial collagen contributes directly to ventricular stiffness at high and low loads and can influence stress-free state and myofiber architecture.

Abstract —Aldosterone upregulates the Na + -K + pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na + -K + pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na + -K + pump current, arising from the 3:2 Na + :K + exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na + in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na + . Aldosterone had no effect on myocardial Na + -K + pump concentration evaluated by vanadate-facilitated [ 3 H]ouabain binding or by K + -dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na + activity. The aldosterone-induced decrease in pump current and increased intracellular Na + were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na + affinity of the Na + -K + pump, whereas it has no effect on maximal pump capacity.

Abstract —A growing body of evidence has been accumulated recently suggesting that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) affect cardiac function, but their mechanism(s) of action is unclear. In the present study, GH and IGF-1 were administered to isolated isovolumic aequorin-loaded rat whole hearts and ferret papillary muscles. Although GH had no effect on the indices of cardiac function, IGF-1 increased isovolumic developed pressure by 24% above baseline. The aequorin transients were abbreviated and demonstrated decreased amplitude. The positive inotropic effects of IGF-1 were not associated with increased intracellular Ca 2+ availability to the contractile machinery but to a significant increase of myofilament Ca 2+ sensitivity. Accordingly, the Ca 2+ -force relationship obtained under steady-state conditions in tetanized muscle was shifted significantly to the left (EC 50 , 0.44±0.02 versus 0.52±0.03 μmol/L with and without IGF-1 in the perfusate, respectively; P 2+ -activated tetanic pressure was increased significantly by 12% (211±3 versus 235±2 mm Hg in controls and IGF-1–treated hearts, respectively; P i or high-energy phosphate content, as assessed by 31 P nuclear magnetic resonance spectroscopy, and were blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Concomitant administration of IGF binding protein-3 blocked IGF-1–positive inotropic action in ferret papillary muscles. In conclusion, IGF-1 is an endogenous peptide that through a wortmannin-sensitive pathway displays distinct positive inotropic properties by sensitizing the myofilaments to Ca 2+ without increasing myocyte [Ca 2+ ] i .

Abstract —Tumor necrosis factor-α (TNF-α) is elevated in the failing heart. Very little is known about regulation of TNF-α in cardiomyocytes. TNF-α expression by macrophages is diminished by adenosine. Therefore, we hypothesized that a similar mechanism might occur in the heart. Neonatal rat myocytes were stimulated with lipopolysaccharide (LPS), and TNF-α was measured by ELISA. In the absence of LPS, myocytes did not release TNF-α in the medium. After exposure to LPS, TNF-α increased to 70.1±3.5 pg/mL at 6 hours. Immunofluorescent staining confirmed that TNF-α was expressed in myocytes. Adenosine decreased TNF-α in a dose-dependent manner (1 to 100 μmol/L, 37% to 65% decrease, P P 2 agonist PD-125944 (DPMA), by cAMP agonists 8-bromo-cAMP, forskolin, and Ro 20–1724, but not by A 1 and A 3 agonists. Conversely, the effect of adenosine could be suppressed by the adenylate cyclase inhibitor MDL-12,330. Adenosine also inhibited TNF-α in adult rat ventricular myocytes (−60%, P P P P 2 receptor and transduced through a G protein–adenylyl cyclase pathway. These results may explain some cardioprotective effects of adenosine and provide a novel pharmacological intervention in congestive heart failure.

Abstract —Dystrophin and β-dystroglycan are components of a complex of at least nine proteins (the dystrophin-glycoprotein complex) that physically link the membrane cytoskeleton in skeletal and cardiac muscle, through the plasma membrane, to the extracellular matrix. Mutations in the dystrophin gene, which result in an absence or a quantitative or qualitative alteration of dystrophin, cause a subset of familial dilated cardiomyopathies as well as Duchenne and Becker muscular dystrophy. Biochemical studies on isolated skeletal muscle molecules indicate that dystrophin is bound to the glycoprotein complex via β-dystroglycan, with the C-terminus of β-dystroglycan binding to the cysteine-rich domain and first half of the C-terminal domain of dystrophin. Ultrastructural labeling has demonstrated a close spatial relationship between dystrophin and β-dystroglycan in intact skeletal muscle, but no previous ultrastructural labeling studies have examined the dystrophin/β-dystroglycan interaction in cardiac muscle. In the present study, we have applied complementary immunoconfocal microscopy and double immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution visualization of labeled membrane components in thin section and in platinum-carbon replicas, to investigate the spatial relationship between dystrophin and β-dystroglycan in rat cardiac muscle. When immunogold probes of two different sizes for the two proteins were used, “doublets” representing side-by-side antibody labeling were demonstrated in en face views at the level of the plasma membrane. The results support the conclusions that dystrophin and β-dystroglycan directly interact at the cytoplasmic face of the rat cardiac muscle plasma membrane.

It is not clear how mitochondrial energy production is regulated in intact tissue when energy consumption suddenly changes. Whereas mitochondrial [NADH] ([NADH] m ) may regulate cellular respiration rate and energetic state, it is not clear how [NADH] m itself is controlled during increased work in vivo. We have varied work and [Ca 2+ ] in intact cardiac muscle while assessing [NADH] m using fluorescence spectroscopy. When increased work was accompanied by increasing average [Ca 2+ ] c (by increasing [Ca 2+ ] o or pacing frequency), [NADH] m initially fell and subsequently recovered to a new steady state level. Upon reduction of work, [NADH] m overshot and then returned to control levels. In contrast, when work was increased without increasing average [Ca 2+ ] c (by increasing sarcomere length), [NADH] m fell similarly, but no recovery or overshoot was observed. This Ca 2+ -dependent recovery and overshoot may be attributed to Ca 2+ -dependent stimulation of mitochondrial dehydrogenases. We conclude that the immediate initial increase in respiration rate upon elevation of work is not activated by increased [NADH] m (since [NADH] m rapidly fell) or by [Ca 2+ ] c (since work could also be increased at constant [Ca 2+ ] c ). However, during sustained high work, a Ca 2+ -dependent mechanism causes slow recovery of [NADH] m toward control values. This demonstrates a Ca 2+ -dependent feed-forward control mechanism of cellular energetics in cardiac muscle during increased work.

Abstract l -Methionine is an essential amino acid that has been reported to have a potent positive inotropic effect on the mammalian myocardium. We studied the mechanisms of the inotropic effect in ventricular myocardium from the rabbit. In the isolated coronary-perfused whole heart, l -methionine in a millimolar range exerted concentration-dependent positive inotropic effects on the isovolumic left ventricle, which were associated with negative lusitropic effects (prolonged time course of relaxation). The chronotropic state and the coronary perfusion pressure were not affected. These complex effects on the isolated whole heart were not blocked by pretreatment with (μmol/L) propranolol 1, prazosin 1, carbachol 3, staurosporine 1, or [Ser 1 ,Ile 8 ]angiotensin II 0.1. To further study the subcellular mechanisms, isolated ventricular papillary muscles from the same species were loaded with a bioluminescent indicator, aequorin, to monitor [Ca 2+ ] i . In the presence of 3 mmol/L l -methionine, the isometric tension showed a similar combination of the positive inotropic and negative lusitropic effects as observed in the whole heart. In contrast, the simultaneously recorded intracellular Ca 2+ signals did not increase in amplitude but instead decreased. The [Ca 2+ ] i -tension relation shifted to the left compared with that obtained in response to [Ca 2+ ] o . In saponin (250 μg/mL)–treated skinned preparations, 3 mmol/L l -methionine also shifted the force-pCa curve to the left by 0.16 pCa units. This is the first demonstration that an essential amino acid directly acts on the myofilaments and modulates their responsiveness to Ca 2+ , thereby producing a positive inotropic effect.

There is evidence that the failing human left ventricle in vivo subjected to additional preload is unable to use the Frank-Starling mechanism. The present study compared the force-tension relation in human nonfailing and terminally failing (heart transplants required because of dilated cardiomyopathy) myocardium. Isometric force of contraction of electrically driven left ventricular papillary muscle strips was studied under various preload conditions (2 to 20 mN). To investigate the influence of inotropic stimulation, the force-tension relation was studied in the presence of the cardiac glycoside ouabain. In skinned-fiber preparations of the left ventricle, developed tension was measured after stretching the preparations to 150% of the resting length. To evaluate the length-dependent activation of cardiac myofibrils by Ca2+ in failing and nonfailing myocardium, the tension-Ca2+ relations were also measured. After an increase of preload, the force of contraction gradually increased in nonfailing myocardium but was unchanged in failing myocardium. There were no differences in resting tension, muscle length, or cross-sectional area of the muscles between both groups. Pretreatment with ouabain (0.02 mumol/L) restored the force-tension relation in failing myocardium and preserved the force-tension relation in nonfailing tissue. In skinned-fiber preparations of the same hearts, developed tension increased significantly after stretching only in preparations from nonfailing but not from failing myocardium. The Ca2+ sensitivity of skinned fibers was significantly higher in failing myocardium (EC50, 1.0; 95% confidence limit, 0.88 to 1.21 mumol/L) compared with nonfailing myocardium (EC50, 1.7; 95% confidence limit, 1.55 to 1.86 mumol/L). After increasing the fiber length by stretching, a significant increase in the sensitivity of the myofibrils to Ca2+ was observed in nonfailing but not in failing myocardium. These experiments provide evidence for an impaired force-tension relation in failing human myocardium. On the subcellular level, this phenomenon might be explained by a failure of the myofibrils to increase the Ca2+ sensitivity after an increase of the sarcomere length.

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle.

Intracellular free calcium concentration ([Ca2+]i) was measured in isolated ferret ventricular papillary muscles during and after long exposures to ischemia. All experiments were performed at 37 degrees C, and the muscles were stimulated at 1 Hz. Ischemia was simulated by changing from superfusion with oxygenated Tyrode's solution to superfusion with water-saturated gas (95% N2-5% CO2), thus simultaneously stopping oxygenation and restricting the extracellular space. [Ca2+]i was measured with aequorin, which was microinjected into superficial cells of the preparation. Exposure to ischemia caused a complex series of changes in [Ca2+]i. In the first few minutes the changes in [Ca2+]i were variable; however, after approximately 5 minutes all preparations exhibited a progressive increase in amplitude and duration of the stimulated rise in [Ca2+]i (the calcium transient). The amplitude of the calcium transients peaked after approximately 18 minutes of ischemia, when they were 339% of the control value. After this peak, the calcium transients progressively failed to occur in response to stimulation and declined in amplitude; simultaneously, spontaneous oscillations of [Ca2+]i appeared and increased in size and frequency. The oscillations in turn then gradually became less frequent until a large, prolonged (5-10 minute) increase in [Ca2+]i occurred, after which [Ca2+]i returned to a low level. There were no further oscillations after this event, which was seen on average after 37 minutes of ischemia. A slowly progressive contracture often began to develop at about this time. A gradual rise in resting [Ca2+]i occurred during the remainder of the exposure to ischemia. When muscles were reperfused after long exposures to ischemia, there was a very large and prolonged increase in [Ca2+]i, which was usually associated with a contracture and failure of recovery of developed tension. The large increase in [Ca2+]i could be reduced by the inclusion of 3 mM nickel chloride in the reperfusing solution. Comparison between reperfusion with O2 gas versus reperfusion with anoxic Tyrode's solution indicated that reoxygenation was more beneficial to the muscle than resumption of bulk flow. These results reveal the complex spectrum of changes in [Ca2+]i that occur during ischemia and on reperfusion. These changes in [Ca2+]i are likely to play an important role in the generation of ischemic arrhythmias and muscle damage.

Rationale: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction. Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI PKC-P ) expressing mutant cTnI (S43E, S45E, T144E). Methods and Results: Two-dimensional gel analysis showed 7.2±0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model–based analysis for integrative interpretation. Compared to wild-type mice, cTnI PKC-P mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F max ). Additionally, cTnI PKC-P mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI PKC-P mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI PKC-P mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state. Conclusions: A modest increase in PKC-induced cTnI phosphorylation (≈7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).

The influence of endothelin 1 on intracellular Ca2+ transients and isometric contractions was investigated in ferret papillary muscles loaded with the Ca(2+)-regulated bioluminescent indicator aequorin. In concentrations of 3 x 10(-9) to 1 x 10(-7) M, endothelin produced dose-dependent increases in the amplitudes of both aequorin light signals (maximum, 31 +/- 12%) and developed tension (maximum, 64 +/- 13%). The peak aequorin light [( Ca2+]i)-peak tension curve generated by increasing endothelin concentrations was steeper and shifted to the left of the curve generated by varying [Ca2+]o; however, the maximum developed tension produced by endothelin did not exceed that produced by 6 mM [Ca2+]o. The effect of endothelin on the amplitude of the aequorin light signal was less than the effect of [Ca2+]o for similar levels of tension development. Moreover, 1 x 10(-7) M endothelin caused an upward shift in the peak aequorin light-peak tension curve generated by varying [Ca2+]o and increased the maximum twitch force by about 12%. The contractions were prolonged, whereas the time course of the Ca2+ transient was not changed in the presence of endothelin. When the function of the sarcoplasmic reticulum was inhibited by 6 microM ryanodine, 10(-7) M endothelin still increased the force generation without increasing the intracellular peak Ca2+, either during isometric twitches or during tetani.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the mechanism of impaired myocardial function after long-term pressure overload, we studied cardiac muscle mechanical contraction and intracellular calcium transients using the bioluminescent indicator aequorin. Left ventricular papillary muscle preparations were examined from three groups of rats: 1) aging spontaneously hypertensive rats (SHR) with clinical and pathological evidence suggesting heart failure (SHR-F group), 2) age-matched SHRs with no evidence of heart failure (SHR-NF group), and 3) age-matched normotensive Wistar-Kyoto rats (WKY group). Isometric force development was depressed in both SHR groups relative to the WKY group. Resting [Ca2+]i was lower in the SHR-F group, and the time to peak [Ca2+]i was prolonged in this group. The relative increases in peak [Ca2+]i with the inotropic interventions of increased [Ca2+]o and the addition of isoproterenol were similar among groups. Although inotropy increased in all groups with increased [Ca2+]o, after isoproterenol, inotropy increased only in the WKY group. Thus, in SHR myocardium, [Ca2+]i increased after isoproterenol, but inotropy failed to increase. Myosin isozymes were shifted toward the V3 isoform in both SHR groups; the V3 isoform was virtually 100% in papillary muscles from the SHR-F group. These changes may reflect events directly contributing to the development of heart failure or represent adaptive changes to chronic pressure overload and heart failure.

In rabbit papillary muscle contracting at 20 degrees C in nitrogen at 0.2 Hz, glycolytic ATP formation is just enough to support the diminished contractile activity. Basal metabolism, important to maintain cellular function and integrity, is strongly inhibited. In the present study, we address the question of whether the inhibition of basal processes in hypoxia determines redevelopment of force in reoxygenation. By not stimulating the muscle during hypoxia, we try to make more ATP available for basal processes. Isometric force of papillary muscles (0.2-Hz stimulation) is measured before, during, and after 40 minutes of hypoxia. ATP formation and utilization in hypoxia are estimated from lactate production and changes in nucleotides and creatine compounds. After reoxygenation, muscles stimulated during hypoxia produce a steady-state force of 78% of the aerobic control; resting muscles recover to 94%. In contrast to expectation, lactate production in hypoxic resting muscles is only 30% of that in contracting ones. The findings indicate that basal metabolic rate of hypoxic muscles at rest is 14% of that of quiescent, well-oxygenated myocardium. We conclude that in hypoxic myocardium little ATP is available for basal metabolism, irrespective of the energy demand of the contractile system. It is therefore unlikely that the lower force found after reoxygenation in muscles stimulated during hypoxia is related to the degree of inhibition of basal processes.

It has been claimed that the mechanical performance and the related energy turnover of the left ventricle can be reliably predicted on the basis of its time-varying elastance behavior. In its most elementary form, this behavior can be mathematically described by E(t) = P(t)/[V(t)-Vd], where E is ventricular elastance, t is time, P is ventricular pressure, V is ventricular volume, and Vd is the intercept of the end-systolic pressure-volume line on the volume axis. To find out how this behavior of the ventricle as a whole is related to the properties of the myocardium, we tested the energetic prediction for the ventricle that the pressure-volume area of an isovolumic contraction equals the energy released in relaxation in experiments on isolated rabbit papillary muscle at 20 degrees C. To that end, the energy (joules) contained by the force-length area of the muscles, contracting isometrically, was compared with the heat (joules) liberated in relaxation as measured with thermopiles. Mechanical performance of the muscles was varied by altering initial muscle length and external calcium. The slope of the resulting relation between force-length area and heat liberated in relaxation (n = 26) was not significantly different from unity. Thus, the energetic prediction of the time-varying elastance model developed for the whole left ventricle was confirmed by experiments on rabbit papillary muscle at 20 degrees C.

When applied extracellularly in the micromolar range, ATP and related compounds induced a positive inotropy in the rat papillary muscle. This was also true in the rat auricle after pertussis toxin treatment. Then, in both tissues, ATP further increased the contraction after a maximal beta-adrenergic stimulation. The increase in contractile force could be related to the increase in the calcium current. The L-type calcium current was measured by whole-cell patch-clamp recording in single cells isolated from the rat ventricle after the sodium and potassium currents were inhibited by tetrodotoxin and cesium, respectively. When added alone, 10 microM ATP increased the calcium current by 60%. Adenosine 5'-O-(3-thiotriphosphate) was also able to increase calcium current. Adenosine was much less effective, and GTP, UTP, CTP, and ITP were without effect. A similar increase in calcium current was observed when ATP was added in addition to a maximal stimulation by a beta-adrenergic agonist or after internal perfusion with cyclic AMP. However, this increase was preceded by a transient decrease whose origin could not be attributed to a P1-purinergic agonistic effect of ATP. The transient decrease was not elicited by adenosine or in a magnesium-free HEPES solution and was not suppressed after pertussis toxin treatment. This effect appeared related to the variations in the holding current also observed upon ATP application. Together with vasodilation, ATP and adenine compounds induced positive inotropy. The latter effect could be attributed in part to the increase in calcium current and was independent of cyclic AMP. Both effects are complementary with the beta-adrenergic stimulation and can help healthy cells to compensate the failing zone from which ATP could be released.

The mechanism of twitch prolongation of hypoxic myocardium after reoxygenation was studied before and after interventions that affect cellular cyclic nucleotide levels, subcellular calcium handling, or oxygen-derived free radical production/survival. Right ventricular ferret papillary muscles were subjected to two 20-minute periods of hypoxia, each followed by 1 hour of reoxygenation. The first sequence of hypoxia/reoxygenation was done without intervention. Before the second sequence, the pharmacological agent under study was added to the superfusate or the superfusate calcium concentration was increased from 2.5 to 8 mM. Time from peak to 80% decline in twitch tension was measured in the presence and absence of each intervention immediately before each period of hypoxia and after reoxygenation at maximal twitch prolongation. Interventions that affect Ca2+ flux across the sarcolemma (verapamil and 8 mM [Ca2+]o) or agents that affect oxygen free radical production/survival (dimethyl sulfoxide and allopurinol) did not affect twitch prolongation. Pharmacological agents that increase cyclic AMP levels (forskolin and milrinone) or those that inhibit mitochondrial activity (oligomycin B and ruthenium red) attenuated twitch prolongation. Pharmacological agents that decrease cyclic AMP levels (carbachol) or inhibit function of the sarcoplasmic reticulum (ryanodine) augmented twitch prolongation. The effect of mitochondrial inhibitors on intracellular calcium handling during hypoxia and reoxygenation was examined using muscles loaded with the bioluminescent calcium indicator aequorin. Mitochondrial inhibitors abbreviated the calcium transient and maximal twitch prolongation after hypoxia. We conclude that alterations in sarcoplasmic reticulum and mitochondria calcium handling contribute to the prolonged relaxation seen upon reoxygenation of hypoxic myocardium.

Atrial fibrillation (AF) may result from stationary reentry in the left atrium (LA), with fibrillatory conduction toward the right atrium (RA). We hypothesize that periodic input to the RA at an exceedingly high frequency results in disorganized wave propagation, compatible with fibrillatory conduction. Simultaneous endocardial and epicardial optical mapping (di-4-ANEPPS) was performed in isolated, coronary-perfused sheep RA. Rhythmic pacing of Bachmann’s bundle allowed well-controlled and realistic conditions for LA-driven RA. Pacing at increasingly higher frequencies (2.0 to 6.0 Hz) led to increasing delays in activation distal to major branching sites of the crista terminalis and pectinate bundles, culminating in spatially distributed intermittent blockade at or above ≈6.5 Hz. At this “breakdown frequency,” the direction of RA propagation became completely variable from beat to beat and thus transformed into fibrillatory conduction. Such frequency-dependent changes were independent of action potential duration. Rather, the spatial boundaries between proximal and distal frequencies correlated well with branch sites of the pectinate musculature. Thus, there exists a breakdown frequency in the sheep RA below which activity is periodic throughout the atrium and above which it is fibrillation-like. The data are consistent with the ideas that during AF, high-frequency activation initiated in the LA undergoes fibrillatory conduction toward the RA, and that sink-to-source effect at branch points of the crista terminalis and pectinate muscles is important in determining the complexity of the arrhythmia.

The reversibility of ischemia-induced changes of extracellular K + concentration ([K + ] o ), resting membrane potential (E M ), and passive cable-like properties, ie, extracellular resistance and cell-to-cell electrical coupling, and their relationship to recovery of conduction and contraction is described in 25 reperfused rabbit papillary muscles. No-flow ischemia caused extracellular K + accumulation, depolarization of E M , an increase in whole-tissue (r t ), external (r o ), and internal (r i ) longitudinal resistances, and failure of conduction and contraction. Muscles were reperfused 10 minutes after the onset of ischemia related cell-to-cell electrical uncoupling, ie, 26±1 minutes after arrest of perfusion. In 11 muscles, incomplete reflow occurred with only partial recovery of [K + ] o and r t . In the remaining 14 muscles, reperfusion caused a rapid and parallel decrease in [K + ] o , r t , and r o . When complete tissue reperfusion occurred, cell-to-cell electrical uncoupling was largely reversible. Thus, cell-to-cell electrical uncoupling did not indicate irreversible injury. Reperfusion induced a depolarizing current widening the difference between the K + equilibrium potential and the E M . This difference decreased after longer periods of reperfusion. Conduction was restored and conduction velocity approached preischemic values as cell-to-cell electrical interaction was reestablished and E M recovered. The recovery of r o preceded r i , decreasing the ratio of the extracellular to intracellular resistance early in reperfusion, an effect predicted to influence the amplitude of the extracellular voltage field and electrocardiographic ST segments during reperfusion.

This study was designed to gain additional insight into the mechanism of the slow force response (SFR) to stretch of cardiac muscle. SFR and changes in intracellular Na+ concentration ([Na+]i) were assessed in cat papillary muscles stretched from 92% to ≈98% of Lmax. The SFR was 120±0.6% (n=5) of the rapid initial phase and coincided with an increase in [Na+]i. The SFR was markedly depressed by Na+-H+ exchanger inhibition, AT1 receptor blockade, nonselective endothelin-receptor blockade and selective ETA-receptor blockade, extracellular Na+ removal and inhibition of the reverse mode of the Na+-Ca2+ exchange by KB-R7943. KB-R7943 prevented the SFR but not the increase in [Na+]i. Inhibition of endothelin-converting enzyme activity by phosphoramidon suppressed both the SFR and the increase in [Na+]i. The SFR and the increase in [Na+]i after stretch were both present in muscles with their endothelium (vascular and endocardial) made functionally inactive by Triton X-100. In these muscles, phosphoramidon also suppressed the SFR and the increase in [Na+]i. The data provide evidence that the last step of the autocrine-paracrine mechanism leading to the SFR to stretch is Ca2+ entry through the reverse mode of Na+-Ca2+ exchange., Facultad de Ciencias Médicas, Centro de Investigaciones Cardiovasculares

Abstract —To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V max of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K m for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F max ), rate of tension redevelopment ( k tr ), and unloaded shortening velocity ( V u ) in trabeculae from both untreated and PTU-treated rats (at maximal Ca 2+ activation), and F-actin sliding speed in an in vitro motility assay ( V f ). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F max is unchanged even though k tr , V u , and V f are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.

Experiments were performed in isolated cat papillary muscles loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in the esterified form to study the effect of endothelin-1 (ET-1) on the activity of the Na+-independent Cl−-HCO3− exchanger. Exposure to ET-1 (10 nmol/L) raised pHi by 0.13±0.03 U (P3−-free solution, whereas no significant change was detected under CO2/HCO3−-buffered medium. However, if ET-1 was applied to muscles pretreated with the anion exchanger inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid, pHi increased by 0.09±0.02 U (P2/HCO3− buffer. The rate of pHi recovery from trimethylamine hydrochloride–induced intracellular alkaline load was enhanced so that net HCO3 efflux increased about three times in the presence of ET-1 (2.74±0.25 versus 9.66±1.29 mmol · L−1 · min−1 at pHi 7.55, PA receptors). BQ 123 also abolished angiotensin II–induced activation of the Na+ independent Cl−-HCO3− exchanger. These results show that ET-1 increases the activity of the Na+-independent Cl−-HCO3− exchanger in cardiac tissue through the ETA receptors. Furthermore, our data suggest that the previously described angiotensin II–induced stimulation of the anion exchanger activity is mediated by endogenous ET-1., Facultad de Ciencias Médicas

Abstract —A mutation in the cardiac β-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the α-myosin heavy chain (αMHC 403/+ ). Myofibrillar disarray with variable penetrance occurred in the left ventricular free wall of the αMHC 403/+ hearts. In resting strips (pCa 8), dynamic stiffness was ≈40% greater than in wild-type strips, consistent with elevated diastolic stiffness reported for murine hearts with FHC. At pCa 6 (submaximal activation), strip isometric tension was ≈3 times higher than for wild-type strips, whereas at pCa 5 (maximal activation), tension was marginally lower. At submaximal calcium activation the characteristic frequencies of the work-producing ( b ) and work-absorbing ( c ) steps of the crossbridge were less in αMHC 403/+ strips than in wild-type strips ( b =11±1 versus 15±1 Hz; c = 58±3 versus 66±3 Hz; 27°C). At maximal calcium activation, strip oscillatory power was reduced (0.53±0.25 versus 1.03±0.18 mW/mm 3 ; 27°C), which is partly attributable to the reduced frequency b , at which crossbridge work is maximum. The results are consistent with the hypothesis that the R403Q mutation reduces the strong binding affinity of myosin for actin. Myosin heads may accumulate in a preforce state that promotes cooperative activation of the thin filament at submaximal calcium but blunts maximal tension and oscillatory power output at maximal calcium. The calcium-dependent effect of the mutation (whether facilitating or debilitating), together with a variable degree of fibrosis and myofibrillar disorder, may contribute to the diversity of clinical symptoms observed in murine FHC.

Abstract —The purpose of this study was to determine the spatial changes in the transmembrane potential caused by extracellular electric field stimulation. The transmembrane potential was recorded in 10 guinea pig papillary muscles in a tissue bath using a double-barrel microelectrode. After 20 S1 stimuli, a 10-ms square wave S2 shock field with a 30-ms S1–S2 coupling interval was given via patch shock electrodes 1 cm on either side of the tissue during the action potential plateau. Two shock strengths (2.1±0.2 and 6.5±0.6 V/cm) were tested with both shock polarities. The recording site was moved across the tissue along fibers with either 200 μm (macroscopic group [n=5], 12 consecutive recording sites over a 2.2-mm tissue length in each muscle) or 20 μm (microscopic group [n=5], 21 consecutive recording sites over a 0.4-mm tissue length in each muscle) between adjacent recording sites. In the macroscopic group, the portion of the tissue toward the anode was hyperpolarized, whereas the portion toward the cathode was depolarized, with 1 zero-potential crossing from hyperpolarization to depolarization present near the center of the tissue. In the microscopic group, only 1 zero-potential crossing was observed in the center region of the tissue, whereas, away from the center, only hyperpolarization was observed toward the anode and depolarization toward the cathode. Although these results are consistent with predictions from field stimulation of continuous representations of myocardial structure, ie, the bidomain and cable equation models, they are not consistent with the prediction of depolarization-hyperpolarization oscillation from representations based on cellular-level resistive discontinuities associated with gap junctions, ie, the sawtooth model.

During myocardial ischemia, electrical uncoupling and contracture herald irreversible damage. In the present study, we tested the hypothesis that an increase of intracellular Ca 2+ is an important factor initiating these events. Therefore, we simultaneously determined tissue resistance, mechanical activity, pH o , and intracellular Ca 2+ (with the fluorescent indicator indo 1, Molecular Probes, Inc) in arterially perfused rabbit papillary muscles. Sustained ischemia was induced in three experimental groups: (1) control, (2) preparations preconditioned with two 5-minute periods of ischemia followed by reperfusion, and (3) preparations pretreated with 1 mmol/L iodoacetate to block anaerobic metabolism and minimize acidification during ischemia. In a fourth experimental group, intracellular Ca 2+ was increased under nonischemic conditions by perfusing with 0.1 mmol/L ionomycin and 0.1 μmol/L gramicidin. Ca 2+ transients and contractions rapidly disappeared after the induction of ischemia. In the control group, diastolic Ca 2+ began to rise after 12.6±1.3 minutes of ischemia; uncoupling, after 14.5±1.2 minutes of ischemia; and contracture, after 12.6±1.5 minutes of ischemia (mean±SEM). Preconditioning significantly postponed Ca 2+ rise, uncoupling, and contracture (21.5±4.0, 24.0±4.1, and 23.0±5.3 minutes of ischemia, respectively). Pretreatment with iodoacetate significantly advanced these events (1.9±0.7, 3.6±0.9, and 1.9±0.2 minutes of ischemia, respectively). In all groups, the onset of uncoupling always followed the start of Ca 2+ rise, whereas the start of contracture was not different from the rise in Ca 2+ . Perfusion with ionomycin and gramicidin permitted estimation of a threshold [Ca 2+ ] for electrical uncoupling of 685±85 nmol/L. In conclusion, the rise in intracellular Ca 2+ is the main trigger for cellular uncoupling during ischemia. Contracture is closely associated with the increase of intracellular Ca 2+ during ischemia.

Abstract To elucidate the mechanisms controlling pH i in myocardium of the spontaneously hypertensive rat (SHR), experiments were performed in papillary muscles (isometrically contracting at 0.2 Hz) from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats loaded with the pH-sensitive fluorescent probe BCECF-AM. An enhanced activity of the Na + -H + exchanger was detected in the hypertrophic myocardium of SHR. This conclusion was based on the following: (1) The myocardial pH i was more alkaline in SHR (7.23±0.03) than in WKY rats (7.10±0.03) ( P i in the SHR (pH i 7.26±0.03 and 7.28±0.03 before and after SITS, respectively). (3) The fall in pH i observed after 20 minutes of Na + -H + exchanger inhibition [5 μmol/L 5-( N -ethyl- N -isopropyl)amiloride (EIPA)] was greater in SHR (−0.16±0.01) than in WKY rats (−0.09±0.02, P i recovery from an intracellular acid load was faster in SHR than in WKY rats (0.068±0.02 versus 0.014±0.002 pH units/min at pH i 6.99, P i recovery from the same acid load decreased to a similar value in both rat strains (0.0032±0.002 pH units/min in SHR and 0.0032±0.002 pH units/min in WKY rats). Under the more physiological HCO 3 − -CO 2 buffer, no significant difference in steady state myocardial pH i was detected between rat strains (7.15±0.03 in SHR and 7.11±0.05 in WKY rats). This finding suggested that an acidifying bicarbonate-dependent mechanism was fully compensating for the hyperactivity of the Na + -H + exchanger in SHR. The following pieces of evidence support an enhanced activity of the Na + -independent Cl − -HCO 3 − exchanger as the mechanism accounting for the compensation: (1) SITS (0.1 mmol/L) increased steady state pH i in the presence of HCO 3 − -CO 2 buffer in SHR (+0.08±0.02, P i recovery from an alkaline load was faster in SHR than in WKY rats (0.075±0.028 versus 0.027±0.016 pH units per minute, respectively; P + independent. (4) No difference in the rate of pH i recovery was detected between SHR and WKY rats when the alkaline load was performed after SITS blockade. Comparison of net HCO 3 − efflux at a given pH i suggests that an increased pH i is not the cause of the hyperactivity of the anion exchanger. Since this anion exchanger is not driving Na + , the offset of the increase in pH i induced by the antiport would not prevent an increase in intracellular Na + mediated by the Na + -H + exchanger.

We examined the effect of several nitric oxide (NO) donors, authentic NO gas, and L-arginine in isolated cat and rat papillary muscles. We did not observe significant inotropic effects in response to any NO donor (ie, SPM-5185, C87-3754, and S-nitroso-N-acetylpenicillamine [SNAP]) from 1 nmol/L to 100 mumol/L. Similarly, authentic NO, at concentrations far in excess of those that maximally dilate the coronary vasculature (ie, 500 nmol/L), also failed to exert a detectable inotropic effect in these preparations. However, in the presence of 5 mumol/L norepinephrine, 500 nmol/L NO exerted a 12 +/- 3% decrease in isolated rat papillary muscle contractility (P < .05). Addition of L-arginine up to 25 mmol/L exerted no inotropic effects in isolated rat papillary muscles. However, at 50 mmol/L, L-arginine decreased contractile force by 21 +/- 4% (P < .01). On further examination, the negative inotropic effect of 50 mmol/L L-arginine appeared to be nonspecific, since the inactive stereoisomer, D-arginine, at 50 mmol/L exerted the same effect. Further studies in isolated adult rat cardiac myocytes elicited similar results, in that 50 mmol/L of L- and D-arginine equally decreased contraction amplitude and the underlying cytosolic calcium transient. Moreover, 500 nmol/L of the NO donor SPM-5185 only modestly decreased contraction amplitude or intracellular calcium in isolated rat cardiac myocytes. These results indicate that administration of physiological concentrations of exogenous NO does not acutely depress the inotropic state of the rat or cat heart to a physiologically significant extent.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

This investigation was designed to elucidate the dynamics of the left ventricular (LV) papillary muscles. Miniature tantalum myocardial markers were placed on the tip and base of each papillary muscle in six dogs. Markers were also implanted into the LV myocardium to define two orthogonal equatorial diameters and the long-axis dimension. Two weeks later, after recovery from thoracotomy, markers were visualized by biplane fluoroscopy, and video images were recorded during control conditions, after autonomic blockade, after inotropic stimulation with calcium, after methoxamine infusion (to increase afterload), and after blood volume augmentation (to increase preload). Two days later, radiographic recordings were made before and after occlusion of the left circumflex coronary artery. Computer-aided analysis of the video recordings was used to determine three-dimensional coordinates of the markers. It was found that before circumflex coronary occlusion, the dynamics of both papillary muscles closely mimicked the dynamics of the LV as a whole. The papillary muscles shortened during ejection and lengthened during diastole. Their lengths changed minimally during the isovolumic periods, and this behavior was not altered by any of the interventions except coronary occlusion. During circumflex coronary artery occlusion, the ischemic posterior papillary muscle lengthened during isovolumic contraction and most of ejection and shortened only when LV pressure began to fall. Hence, we believe that previous studies demonstrating papillary muscle lengthening during isovolumic contraction and shortening during isovolumic relaxation may have been confounded by coexistent myocardial ischemia or stunning.

Myocardial ischemia is characterized by a decrease in phosphocreatine (PCr) and Mg(2+)-ATP contents as well as an accumulation of myosin ATPase reaction products (inorganic phosphate [P(i)], protons, and Mg(2+)-ADP). The possibility that these metabolites play a role in rigor tension development was checked in rat ventricular Triton X-100-skinned fibers. Rigor tension was induced by stepwise decreasing [Mg(2+)-ATP] in the presence or in the absence of 12 mmol/L PCr. To mimic the diastolic ionic environment of the myofibrils, [free Ca2+] was set at 100 nmol/L (pCa 7); [free Mg2+], at 1 mmol/L; and ionic strength, at 160 mmol/L. In control conditions (pH 7.1, with no added P(i) or Mg(2+)-ADP), the pMg(2+)-ATP for half-maximal rigor tension (pMg(2+)-ATP50) was 5.07 +/- 0.03 in the presence of PCr. After withdrawal of PCr, the pMg2+)-ATP50 value was shifted toward higher Mg(2+)-ATP values (3.57 +/- 0.03). Addition of 20 mmol/L P(i) shifted the pMg(2+)-ATP50 to 3.71 +/- 0.04 (P < .05) in the absence of PCr and in the opposite direction to 4.98 +/- 0.02 (P < .01) in the presence of PCr. Acidic pH (6.6) strongly increased pMg(2+)-ATP50 in both the absence (3.90 +/- 0.03, P < .001) and presence (5.44 +/- 0.02, P < .001) of PCr. Conversely, Mg(2+)-ADP (250 mumol/L) decreased pMg(2+)-ATP50 to 3.26 +/- 0.06 (P < .001) in the absence of PCr; at pMg(2+)-ATP 4, no rigor tension was observed until PCr concentration was decreased to < 2 mmol/L. At acidic pH, maximal rigor tension was lower by 29% compared with control conditions, whereas in the presence of Mg(2+)-ADP, maximal rigor tension developed to 143% of the control value; P(i) had no effect. The tension-to-stiffness (measured by the quick length-change technique) ratio was lower in rigor (no PCr and pMg(2+)-ATP 6) than during Ca2+ activation in the presence of both PCr and ATP. Compared with control rigor conditions, this parameter was unchanged by Mg(2+)-ADP and decreased by acidic pH, suggesting a proton-induced decrease in the amount of force per crossbridge. In addition to their known effects on active tension, Mg(2+)-ADP and protons affect rigor tension and influence ischemic contracture development. It is concluded that ischemic contracture and increased myocardial stiffness may be mediated by a decreased PCr and local Mg(2+)-ADP accumulation. This emphasizes the importance of myofibrillar creatine kinase activity in preventing ischemic contracture.

Resting heat rate was measured in superfused rabbit papillary muscles at 20 degrees C during 40 minutes of anoxia and subsequent reoxygenation. To reveal the nature of the reactions underlying energy output under such conditions, the data obtained were compared with values predicted from data on chemical change. Before and after the anoxic period, muscles were stimulated at 0.2 Hz, during which time the contraction-related heat rate was measured. During anoxia, muscles were kept at rest or stimulated at 1 Hz. Stimulation was switched off intermittently to determine resting heat rate. Before anoxia, resting heat rate was 8.7 +/- 1.1 (mean +/- SEM) mW.g dry wt-1. During anoxia, it decreased to 38% and 50% of the preanoxic level in resting and stimulated muscles, respectively (P < .05). In resting muscles, heat rate increased with reoxygenation in approximately 10 to 15 minutes to 1.3 times the preanoxic level, whereas this was 3.7 times in stimulated muscles. Resting heat rate returned within 65 (resting muscles) or 150 (stimulated muscles) minutes to the baseline. The ratio of force- and contraction-related heat rate, ie, the economy of contraction, was not different before and after anoxia. We estimated that the heat produced by muscles during anoxia was not different from the heat to be expected from the hydrolysis of creatine phosphate, the breakdown of nucleotides, and the formation of lactate. The overshoot in resting heat during reoxygenation of resting muscles could be accounted for by the resynthesis of the energy store. The much larger overshoot in resting heat of stimulated muscles was due to the contracture. The finding that the economy of contraction was not altered by anoxia and reoxygenation suggests that both sarcoplasmic reticulum Ca(2+)-ATPase and myofibrillar ATPase are depressed by anoxia and that the enhancement of cytosolic calcium transients with reoxygenation, reported in other studies on papillary muscle, results from reduced binding of calcium rather than from enhanced release.

Vascular endothelium has been shown to modify the contractile characteristics of vascular smooth muscle, and endocardial endothelium has been shown to modify the contractile characteristics of adjacent myocardium. In this study, whether vascular endothelium also modifies the contractile characteristics of adjacent myocardium and whether these effects are additive to those of endocardial endothelium were investigated. Rabbit hearts (n = 54) were excised and mounted in a Langendorff preparation. Vascular reactivity was verified by acetylcholine infusion. One group of these hearts had Triton X-100 injected as a bolus into the coronaries to render the vascular endothelium dysfunctional. The other portion served as control hearts. Triton X-100 bolus injection resulted in little or no pathological changes on morphological examination; however, the vasodilatory response to acetylcholine in these hearts was abolished, suggesting vascular endothelial dysfunction. Vascular smooth muscle reactivity was verified in Triton X-100-injected hearts by nitroprusside infusion. In the control Langendorff-perfused hearts, there was little evidence of vascular endothelial dysfunction, with the coronary perfusion rate increasing from 8.9 +/- 0.4 to 11.0 +/- 0.3 ml/g per minute (p < 0.01) in response to acetylcholine. All hearts were then removed, and right ventricular papillary muscles were excised for myocardial mechanical studies. Control Langendorff-perfused hearts had myocardial mechanical characteristics similar to those of muscles from 18 other control hearts without Langendorff perfusion, indicating that the Langendorff perfusion itself had little effect on myocardial mechanics. The muscles from the Triton X-100-injected Langendorff hearts had marked changes: a shortening of twitch duration (363 +/- 16 versus 449 +/- 9 msec, p < 0.01) and decreases in total tension (2.2 +/- 0.2 versus 2.9 +/- 0.2 g/mm2, p < 0.01), dT/dt (9 +/- 1 versus 12 +/- 1 g/mm2 per second, p < 0.05), and maximum velocity of unloaded muscle shortening (Vmax) (0.89 +/- 0.06 versus 1.14 +/- 0.07 length at which maximum developed tension occurred [Lmax]/sec, p < 0.05). Endocardial endothelial removal of the papillary muscles in the two control groups (with and without Langendorff perfusion) by Triton X-100 caused the same changes in twitch characteristics as occurred in muscles from the Langendorff-perfused hearts injected with Triton X-100 but with intact endocardial endothelium, suggesting that vascular endothelial dysfunction had similar effects on contractile characteristics as endocardial endothelial removal.(ABSTRACT TRUNCATED AT 400 WORDS)

The mechanisms underlying the marked increase in [K+]o in response to ischemia are not fully understood. Accordingly, the present study was performed to assess the contribution of ATP-regulated K+ channels by using simultaneous measurements of cellular K+ efflux, [K+]o, transmembrane action potentials, and tissue ATP, ADP, phosphocreatine, and creatine content in a unique isolated, blood-perfused papillary muscle preparation during hypoxia compared with ischemia. During 15 minutes of hypoxic perfusion (PO2, 6.1 +/- 0.9 mm Hg) with normal [K+]o of 4.1 +/- 0.1 mM, action potential duration (APD) was not altered even though tissue ATP levels decreased markedly from 33.5 +/- 1.8 to 14.7 +/- 2.0 nmol.mg protein-1 (p < 0.01). Net cellular K+ efflux, based on measured differences of [K+] between the venous effluent and the perfusate, was 13.23 +/- 0.79 mumol.g wet wt-1 during hypoxia. In contrast, after 15 minutes of zero-flow ischemia, APD at 80% of repolarization (APD80) decreased by 47% from 171 +/- 5 to 92 +/- 5 msec (p < 0.01), but integrated net cellular K+ efflux over 15 minutes of ischemia was 8.4-fold less (1.57 +/- 0.13 mumol.g wet wt-1) than during hypoxia. Tissue ATP levels, however, decreased by only 35.2% to 21.7 +/- 2.1 nmol.mg protein-1, which was significantly less than that induced by 15 minutes of hypoxia. Perfusion with hypoxic blood containing high [K+]o of 10.3 +/- 0.3 mM resulted in APD shortening similar to that observed during ischemia. Cellular K+ loss, however, was inhibited markedly by high [K+]o perfusion (only 4.51 +/- 0.28 mumol.g wet wt-1). Pretreatment with glibenclamide (5 microM), a drug that has been reported to inhibit ATP-regulated K+ channels and accelerate glycolysis in normoxic tissue, partially inhibited cellular K+ efflux during hypoxic perfusion with normal [K+]o (7.35 +/- 0.71 versus 13.23 +/- 0.79 mumol.g wet wt-1, p < 0.01) but had no significant influence on repolarization time or tissue ATP levels. Although glibenclamide partially prevented action potential shortening induced by hypoxic perfusion in the presence of elevated [K+]o, the proportion of cellular K+ efflux reduced by glibenclamide was less (23%) than that observed with glibenclamide in hypoxic perfusion with normal [K+]o (44%).(ABSTRACT TRUNCATED AT 400 WORDS)

To determine the effects of moderate ethanol consumption on the mechanical, biochemical, and structural characteristics of the heart, myocardial mechanical performance, contractile protein enzyme activity, and the number and size of myocytes were measured in male Fischer 344 rats after the ingestion of 30% oral ethanol. Papillary muscles removed from the left ventricle were greater in length, weight, and cross-sectional area than the corresponding muscles from the right side. However, no differences were found between control and ethanol-treated myocardium when either the left or right side was compared separately. Chronic ethanol ingestion resulted in an increase in resting tension in left ventricular muscles, with no alteration in peak developed tension. Moreover, time to peak tension was significantly prolonged, whereas a depression was observed in the peak rate of isometric tension development. Isotonically, left muscles from ethanol-treated rats revealed a prolongation of time to peak shortening and a marked depression in the velocity of shortening at physiological loads. No changes were noted in muscles from the right ventricle. Contractile protein enzyme activity revealed no differences in myofibrillar Mg(2+)-ATPase activity in right and left ventricular myocardium between control and ethanol-treated rats in the presence of EGTA. However, at physiological activating levels of calcium, an upward shift of the myofibrillar Mg(2+)-ATPase activity-calcium curve occurred in left myocardium, whereas a depression in this relation was seen in the right ventricle. As a result of chronic ethanol intake, a decrease was noted in the volume percent of myocardium occupied by myocytes, and that myocyte cell volume per nucleus was found to remain essentially constant throughout the various layers of the ventricular wall. Importantly, a 14% significant decrease in the total number of myocyte nuclei was demonstrated in the left ventricular myocardium of rats on chronic ethanol consumption. Thus, chronic but moderate alcohol ingestion resulted in depressed contractile performance, alterations in myofibrillar Mg(2+)-ATPase activity, and myocyte loss. These events may serve to function as preliminary indicators of the onset of heart failure of alcoholic origin in this animal model.

The extracellular pH (pHo) and intracellular pH (pHi) were simultaneously measured with H(+)-sensitive microelectrodes in the rabbit papillary muscle during normal arterial perfusion and no-flow ischemia. The preparation was kept in an artificial gaseous atmosphere (N2 and CO2 during ischemia) without a surrounding fluid layer. Cylindrical muscles of small diameters (less than 1.0 mm) were selected to prevent major diffusion gradients of CO2 within the muscle cylinder during ischemia. In normal perfusion with CO2/HCO3(-)-buffered blood at PCO2 of 35 mm Hg, pHi was 7.03 +/- 0.03. During early ischemia, extracellular acidification was much more prominent than intracellular acidification. Consequently, the transmembrane pH gradient reversed (pHo less than pHi) at approximately 8 minutes. At 14 minutes of ischemia, pHo was 6.64 and pHi was 6.93. A moderate increase in PCO2 from 35 to 67 mm Hg before ischemia enhanced intracellular acidification in ischemia. Simulation of CO2 accumulation (increase of PCO2 in the surrounding atmosphere), as encountered in midmural ventricular layers during in vivo ischemia, produced a significant decrease of pHo (6.30 versus 6.64) and pHi (6.65 versus 6.93) at 14 minutes of ischemia. The presence of red blood cells in the intravascular space after arrest of coronary perfusion showed a pronounced effect on extracellular and intracellular acidosis. If the muscles were perfused with CO2/HCO3(-)-buffered perfusate in the absence of red blood cells, the changes of pHo and pHi were significantly larger (pHo, 6.00 versus 6.64; pHi, 6.46 versus 6.93 at 14 minutes) during ischemia. Actively developed force during ischemia was not significantly influenced by conditions modulating pHi. It decreased by 82% after 5 minutes, even when no significant change of pHi was recorded. By contrast, ischemic contracture was dependent on intracellular acidification. It developed earlier in the absence of red blood cells or with low extracellular buffer capacity. It is concluded that during acute myocardial ischemia 1) extracellular acidification exceeds intracellular acidification, 2) the decrease in pHi is inhomogeneous because of local variation in CO2 accumulation and diffusion, 3) the decrease in pHi is relatively small in the presence of red blood cells, and 4) the development of ischemic contracture but not the early decline in active tension is sensitive to changes in pHi.

Based on the premise that short-time-scale, small-amplitude pressure/volume/outflow behavior of the left ventricular chamber was dominated by dynamic processes originating in cardiac myofilaments, a prototype model was built to predict pressure responses to volume perturbations. In the model, chamber pressure was taken to be the product of the number of generators in a pressure-bearing state and their average volumetric distortion, as in the muscle theory of A.F. Huxley, in which force was equal to the number of attached crossbridges and their average lineal distortion. Further, as in the muscle theory, pressure generators were assumed to cycle between two states, the pressure-bearing state and the non-pressure-bearing state. Experiments were performed in the isolated ferret heart, where variable volume decrements (0.01-0.12 ml) were removed at two commanded flow rates (flow clamps, -7 and -14 ml/sec). Pressure responses to volume removals were analyzed. Although the prototype model accounted for most features of the pressure responses, subtle but systematic discrepancies were observed. The presence or absence of flow and the magnitude of flow affected estimates of model parameters. However, estimates of parameters did not differ when the model was fitted to flow clamps with similar magnitudes of flows but different volume changes. Thus, prototype model inadequacies were attributed to misrepresentations of flow-related effects but not of volume-related effects. Based on these discrepancies, an improved model was built that added to the simple two-state cycling scheme, a pathway to a third state. This path was followed only in response to volume change. The improved model eliminated the deficiencies of the prototype model and was adequate in accounting for all observations. Since the template for the improved model was taken from the cycling crossbridge theory of muscle contraction, it was concluded that, in spite of the complexities of geometry, architecture, and regional heterogeneity of function and structure, crossbridge mechanisms dominated the short-time-scale dynamics of left ventricular chamber behavior.

In a previous communication, we showed that beta-adrenergic stimulation of cardiac muscles was associated with an increase in the rate of cycling of crossbridges as measured by perturbation analysis in the frequency domain. In this analysis, the frequency at which dynamic stiffness is a minimum (fmin) is taken as a measure of the rate of crossbridge cycling. In this paper, we test the hypothesis that the beta-adrenergic receptor-induced increase in crossbridge cycling rate is mediated by elevation of the intracellular level of cyclic AMP. The approach taken is to compare the effects on fmin in rat papillary muscles during Ba(2+)-activated contractures of 1) an agonist of cyclic AMP that can easily penetrate the cell, namely, dibutyryl cyclic AMP, 2) agents that block cyclic AMP phosphodiesterase, namely, the xanthine derivatives isobutylmethylxanthine and caffeine, and 3) an inotropic agent that does not affect the intracellular level of cyclic AMP, namely, ouabain. Our results showed that dibutyryl cyclic AMP at a dose of 5 mM has the same actions as beta-adrenergic stimulation: it potentiated the isometric twitch force, reduced the time to peak tension and time to half relaxation, and shifted fmin by a factor of 1.8 +/- 0.1 (n = 5). Isobutylmethylxanthine at up to 1.1 mM also acted in the same manner, increasing fmin by a factor of 1.8 +/- 0.2 (n = 6), but ouabain, at a dose (0.03 mM) sufficient to potentiate twitch force by 40 +/- 2% (n = 4), was without effect on the time course of the twitch nor was fmin changed (n = 4). Our findings support the hypothesis that a beta-adrenergic receptor-mediated increase in crossbridge cycling rate is due to an increase in intracellular cyclic AMP level and illustrate the usefulness of the frequency domain analysis approach in the analysis of the mechanism of action of inotropic agents.

The mechanism by which isoproterenol (ISO) prevents the prolongation of action potential duration (APD) and refractory period (RP) by the class III antiarrhythmic agent E-4031 was studied. E-4031 (1 microM) increased RP by 50% with no effect on contractile force in papillary muscles isolated from guinea pig heart. ISO (1 microM) increased force of contraction more than fivefold and decreased RP by 25%. The prolongation of RP by E-4031 was prevented by pretreatment of muscles with ISO. The prolongation of APD in isolated guinea pig ventricular myocytes by 5 microM E-4031 also was antagonized by prior exposure of the cells to 1 microM ISO. Instantaneous currents and delayed rectifier K+ currents, IK, were measured in isolated myocytes using the suction microelectrode voltage-clamp technique. Currents were measured in response to 225-msec depolarizing pulses from a holding potential of -40 mV. Previous studies have demonstrated that IK in these cells results from activation of two distinct outward K+ currents, IKs and IKr (specifically blocked by E-4031). ISO doubled the magnitude of IKs without significant effect on IKr. The instantaneous current, putatively identified as a Cl- current, also was doubled by ISO but was unaffected by E-4031. The augmented conductance of IKs and instantaneous current by ISO results in a decrease in RP. The small effect of E-4031 on APD and RP in the presence of ISO results from the smaller contribution of IKr relative to the augmented repolarizing currents.

A theoretical model of action potential propagation in a thick strand of cardiac muscle is presented. The calculation takes into account the anisotropic and syncytial properties of the tissue, the presence of the interstitial space, the effect of the surrounding tissue bath, and the variation of the potential both along the strand length and across the strand cross section. The bidomain model is used to represent the electrical properties of the tissue, and the Ebihara-Johnson model is used to represent the properties of the active sodium channels. The calculated wave front is curved, with the action potential at the surface of the strand leading that at the center. The rate of rise of the action potential and the time constant of the action potential foot vary with depth into the tissue. The velocity of the wave front is nearly independent of strand radius for radii greater than 0.5 mm. The conduction velocity decreases as the volume fraction of the interstitial space decreases. In the limit of tightly packed cells, an action potential propagates quickly over the surface of the strand; the bulk of the tissue is then excited by a slow inward wave front initiated on the surface. This model does not predict an increase in conduction velocity when cells are tightly packed, a hypothesis that has been proposed previously to explain the fast conduction velocity in Purkinje fibers of some species.

Acidosis leads to mechanical alternans (i.e., alternation of large and small contractions) in ferret papillary muscles. This alternation in the size of the contraction is paralleled by alternation in the size of the intracellular Ca2+ transient (monitored using the photoprotein aequorin). In isolated myocytes, the large contraction is accompanied by a prolonged action potential. Mechanical alternans also can be induced by acidosis in isolated myocytes during a train of voltage-clamp pulses. Thus, it appears unlikely that the mechanical alternans is secondary to changes in action potential duration; it is more likely that the observed changes in action potential duration are secondary to changes in the size of the Ca2+ transient. The observation that a Ca2(+)-activated inward current also shows alternation during mechanical alternans provides a possible mechanism for the link between Ca2+ and action potential duration. The alternation in the size of the Ca2+ transient may be secondary to the slowed mechanical restitution observed in papillary muscles during acidosis. This also could explain the observation that decreasing stimulation rate can abolish the alternans.

The programmed onset of myocardial dysfunction and its progression to congestive heart failure in the cardiomyopathic Syrian hamster is hallmarked by alterations in myocellular calcium regulation. To determine whether calcium channel blockade is effective in halting the progressive depression of myocardial contractile performance in this animal model of congestive heart failure, oral verapamil therapy was instituted at 50 days of age, and treatment continued for various durations until the time of study at either 150 or 250 days of age. Left ventricular papillary muscle isometric and isotonic performance, as well as transmembrane electrical characteristics, was depressed in diseased hamsters at 150 days of age and deteriorated further by 250 days of age. These changes were evidenced by prolongation of contraction duration, a marked depression in the load-velocity relation, and a significant prolongation in the repolarization phase of the transmembrane action potential. Myocardial functional and electrical alterations associated with the progression of life in myopathic hamsters were completely halted by verapamil therapy that was continuous from 50 days after birth until death by diastolic arrest, at 150 or 250 days of age. However, premature termination of verapamil treatment before death resulted in a progressive renewal of the functional and electrical alterations for the duration of drug termination. It is concluded that the pathological changes seen during the lifetime of the cardiomyopathic hamster can be prevented by continuous calcium channel blockade and that intermediate prevention can be attained by protracted verapamil therapy. Thus, chronic verapamil therapy may be a useful adjunct in the prevention of human congestive heart failure of similar etiology.

We investigated the contribution of opening of the ATP-sensitive K+ channel to extracellular accumulation of K+ during ischemia with the use of glibenclamide, a specific blocker of this K+ channel. To characterize the electrophysiological effects of glibenclamide during metabolic inhibition (by either application of dinitrophenol or hypoxia) we performed patch-clamp studies in isolated membrane patches of guinea pig myocytes and in intact guinea pig myocytes and studied action potential parameters in isolated superfused guinea pig papillary muscle. We studied the effect of glibenclamide on extracellular accumulation of K+ and H+ in isolated retrogradely perfused globally ischemic hearts of rat, guinea pig, and rabbit. Experimental evidence is presented that supports the conclusions that glibenclamide 1) effectively blocks open K+ATP channels, 2) reverses the dinitrophenol-induced increase of the outward current and prevents the hypoxia-induced shortening of the action potential, 3) decreases the rate of K+ accumulation during the first minutes of ischemia in stimulated hearts, an effect which was entirely absent in quiescent hearts, and 4) does not influence the rate and extent of ischemia-induced extracellular acidification.

The relation among passive electrical resistive properties, longitudinal conduction velocity, extracellular potassium concentration, [K+]o, and mechanical activity was investigated in the isolated rabbit papillary muscle during normal arterial perfusion and no-flow ischemia in the presence and absence of verapamil, or a reduced extracellular Ca2+ concentration [Ca2+]o. During normal arterial perfusion, verapamil (0.5 microM, free [Ca2+]o = 1.0 mM) and hypocalcemic blood perfusate (free [Ca2+]o = 0.4 mM) reduced the maximal isometric twitch tension by 48% and 78%, depolarized the resting membrane by +3 and +7 mV, decreased the extracellular longitudinal resistance (ro) by 15% and 26%, and increased conduction velocity by 4% and 6%, respectively. The changes in conduction velocity during these interventions were consistent with those predicted by linear cable theory (+3% and +9%) for the observed changes in ro. In contrast, verapamil shortened whereas a reduced [Ca2+]o lengthened action potential duration. Comparison of simultaneously measured longitudinal whole tissue resistance (rt), intracellular longitudinal resistance (ri), [K+]o, and resting tension during ischemia showed a close association between abrupt cell-to-cell electrical uncoupling, development of ischemic contracture, and the secondary rise of [K+]o, which all started to develop after approximately 15 minutes of ischemia. Electrical cell-to-cell uncoupling was completed within 15 minutes. In the presence of verapamil, the relation among the onset of electrical cell-to-cell uncoupling, secondary rise of [K+]o, and onset of ischemic contracture in ischemia was qualitatively the same as in its absence; however, these events were postponed by approximately 10 minutes, and the rates of contracture development and uncoupling were diminished. Conduction velocity decreased after 12 minutes of ischemia from 54 to 36 cm/sec in the absence of and from 61 to 46 cm/sec in the presence of verapamil. This slowing effect on impulse conduction could not be attributed to changes of electrical cell-to-cell coupling because at this time an increase in ri had not yet taken place. In the presence of a reduced [Ca2+]o, the resting tension and ri increased almost immediately after the onset of ischemia. Although the resting tension rose progressively throughout the course of ischemia, the ri showed a biphasic increase characterized by an early transient increase that reached a peak at 8 minutes (+87%) and a second, irreversible increase beginning at approximately 12 minutes. This final onset of electrical cell-to-cell uncoupling and the secondary rise of [K+]o were not different from the findings with a normal [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)