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The main goal was to investigate the effectiveness of cryosauna in preventing the development of delayed onset muscle soreness and to analyze the regenerative changes within muscles after acute fatigue-induced exercises. Thirty-one volunteers were assigned into two groups: 1) an intervention group that participated in cryostimulation after fatigue-induced exercise protocol (CRYO, n = 16) and a control group that performed fatigue-induced exercise protocol, but without any intervention (CONT, n = 15). Main outcome measures include at baseline: blood sample testing (leukocyte content, myoglobin concentration, and creatine kinase activity) and muscle stiffness of lower extremity; immediately after (stiffness), and 24-48-72-96 h post-exercise (blood samples and stiffness). Both groups performed an exercise-induced muscle damage protocol based on repeated countermovement jumps (10 sets, 10 repetitions). The CRYO group underwent a cryosauna (temperature: -110°C, time: 1.5 min per session) intervention during four sessions (i.e., immediately after, 24-48-72 h post-exercise). Leukocyte content was significantly greater 24-48-72 h after exercise in CONT, compared with the CRYO group ( p ≤ 0.05 for all), while creatine kinase activity was greater 24-48-96 h in CONT, compared with the CRYO group ( p ≤ 0.05 for all). Muscle stiffness increased significantly in rectus femoris, tibialis anterior, and fibula muscle after 48 h post-exercise ( p ≤ 0.05 for all), as well as in tibialis anterior and fibula after 72 h post-exercise ( p ≤ 0.05 for all) in the CRYO group. Multiple cryosauna was an effective recovery strategy that reduced blood biomarkers and muscle stiffness after exercise-induced muscle damage. Moreover, the development of delayed onset muscle soreness, expressed by a greater muscle stiffness post-exercise, was attenuated to the first 48 h., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wolska, Domagała, Kisilewicz, Hassanlouei, Makar, Kawczyński and Klich.)

Bone heat-induced changes complicate osteometric stature estimation of human remains from forensic settings. The validity of current methods has not been tested to a great extent. Our aim was to determine how precise are stature estimations obtained from three different approaches, namely by using (i) Rösing's method (Rösing 1977), (ii) a 10% shrinkage correction factor (Strzałko et al. 1972) and (iii) chemosteometry (Gonçalves et al. 2020). For this purpose, pre- and post-burned head measurements from the humerus, radius and femur were used. The sample comprised 46 human skeletons of known sex and age-at-death. These were experimentally burnt to maximum temperatures ranging from 700 to 1100°C (attained after 90-188 min) for other research purposes. Stature estimations were performed through measurements in both pre-burned and burned bones using the three approaches and based on the method of Olivier and Tissier (1975). Mean absolute differences and the relative technical errors of measurements (TEM%) between the pre-burned and the estimations were calculated. Absolute mean differences indicated that the chemosteometric approach allowed for potentially more precise stature estimations than the other two procedures. However, the TEM% was very low for all approaches (smaller or close to 1%), and stature estimations were thus well within the error margin reported by Olivier and Tissier (1975). Results suggest that the chemosteometric approach enables more accurate predictions of the actual heat-induced shrinkage of each bone thus rendering more precise stature estimations. Nonetheless, the other procedures also provided quite reliable estimations although they require confirmation that the bone is calcined., (© 2022 American Academy of Forensic Sciences.)

Background: Caged drug-eluting stents impede natural coronary rotational motion and increase vessel stress, which can contribute towards adverse events. The DynamX™ Drug-Eluting Bioadaptor is a cobalt‑chromium platform with a novel mechanism that uncages the vessel after the bioresorbable coating resorbs over six months. This study aimed to analyze the effects of the rotational uncaging in a finite element analysis (FEA) model, validating its effect on coronary artery rotational motion through in-vivo stationary intravascular ultrasound (IVUS)., Methods: Maximum Von Mises stresses were measured in an FEA model and compared for caged and uncaged bioadaptors. Stationary IVUS images from 20 patients enrolled in a single center were acquired post implantation and at 9-12-month follow-up to evaluate coronary artery rotational motion., Results: The FEA model showed that rotational uncaging of the bioadaptor reduces peak stress by 70%. In-vivo, the in-bioadaptor segment was significantly distorted post-implant compared to the native distal and proximal vessel, measured by IVUS: The sum of clockwise and counterclockwise rotational motion (net-effect rotational motion) was -2.7 ± 4.3° versus 0.5 ± 5.0° (proximal vessel), p = 0.036, and versus 0.2 ± 3.8° (distal vessel), p = 0.042. At follow up, when the bioadaptor had uncaged, the vessel returned towards its equilibrium (net-effect rotational motion -0.2 ± 5.6°), with no significant difference between the vessel segments., Conclusions: In concurrence with the FEA observation, the in-vivo IVUS-analysis demonstrates that uncaging of the bioadaptor affects coronary artery rotational motion. The effect of these findings on reducing clinical events warrants further investigation., Competing Interests: Declaration of competing interest M.K. reports consulting fees from Elixir Medical, S.V. reports consulting fees from Neovasc outside the submitted work and consulting fees from Elixir Medical. M.V. reports royalty payments from Merit Medical, and research grants from Boston Scientific. All other authors reported no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ΔF/F(0)=3.52±0.38, n=14; Ctrl(15min): ΔF/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (τ: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10μmol/L) and the Akt inhibitor V (20μmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect., (Published by Elsevier Ltd.)

1. Acidosis in cardiac muscle is associated with a decrease in developed force. We hypothesized that slow skeletal troponin I (ssTnI), which is expressed in neonatal hearts, is responsible for the observed decreased response to acidic conditions. To test this hypothesis directly, we used adult transgenic (TG) mice that express ssTnI in the heart. Cardiac TnI (cTnI) was completely replaced by ssTnI either with a FLAG epitope introduced into the N-terminus (TG-ssTnI) or without the epitope (TG-ssTnI) in these mice. TG mice that express cTnI were also generated as a control TG line (TG-cTnI). Non-transgenic (NTG) littermates were used as controls. 2. We measured the force-calcium relationship in all four groups at pH 7.0 and pH 6.5 in detergent-extracted fibre bundles prepared from left ventricular papillary muscles. The force-calcium relationship was identical in fibre bundles from NTG and TG-cTnI mouse hearts, therefore NTG mice served as controls for TG-ssTnIand TG-ssTnI mice. Compared to NTG controls, the force generated by fibre bundles from TG mice expressing ssTnI was more sensitive to Ca(2+). The shift in EC(50) (the concentration of Ca(2+) at which half-maximal force is generated) caused by acidic pH was significantly smaller in fibre bundles isolated from TG hearts compared to those from NTG hearts. However, there was no difference in the force-calcium relationship between hearts from the TG-ssTnIand TG-ssTnI groups. 3. We also isolated papillary muscles from the right ventricle of NTG and TG mouse hearts expressing ssTnI and measured isometric force at extracellular pH 7.33 and pH 6.75. At acidic pH, after an initial decline, twitch force recovered to 60 +/- 3 % (n = 7) in NTG papillary muscles, 98 +/- 2 % (n = 5) in muscles from TG-ssTnIand 96 +/- 3 % (n = 7) in muscles from TG-ssTnI hearts. Our results indicate that TnI isoform composition plays a crucial role in the determination of myocardial force sensitivity to acidosis.

We studied how the nitric oxide (NO*) donor 3-morpholinosydnonimine (SIN-1) alters the response to beta-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO*, are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of beta-adrenergic stimulation.

Activation of protein kinase C (PKC) in heart muscle signals hypertrophy and may also directly affect contractile function. We tested this idea using a transgenic (TG) mouse model in which conditionally expressed PKCbeta was turned on at 10 wk of age and remained on for either 6 or 10 mo. Compared with controls, TG cardiac myocytes demonstrated an increase in the peak amplitude of the Ca(2+) transient, an increase in the extent and rate of shortening, and an increase in the rate of relengthening at both 6 and 10 mo of age. Phospholamban phosphorylation and Ca(2+)-uptake rates of sarcoplasmic reticulum vesicles were the same in TG and control heart preparations. At 10 mo, TG skinned fiber bundles demonstrated the same sensitivity to Ca(2+) as controls, but maximum tension was depressed and there was increased myofilament protein phosphorylation. Our results differ from studies in which PKCbeta was constitutively overexpressed in the heart and in studies that reported a depression of myocyte contraction with no change in the Ca(2+) transient.

We used transgenic (TG) mice overexpressing mutant alpha-tropomyosin [alpha-Tm(Asp175Asn)], linked to familial hypertrophic cardiomyopathy (FHC), to test the hypothesis that this mutation impairs cardiac function by altering the sensitivity of myofilaments to Ca(2+). Left ventricular (LV) pressure was measured in anesthetized nontransgenic (NTG) and TG mice. In control conditions, LV relaxation was 6,970 +/- 297 mmHg/s in NTG and 5,624 +/- 392 mmHg/s in TG mice (P < 0.05). During beta-adrenergic stimulation, the rate of relaxation increased to 8,411 +/- 323 mmHg/s in NTG and to 6,080 +/- 413 mmHg/s in TG mice (P < 0.05). We measured the pCa-force relationship (pCa = -log [Ca(2+)]) in skinned fiber bundles from LV papillary muscles of NTG and TG hearts. In control conditions, the Ca(2+) concentration producing 50% maximal force (pCa(50)) was 5.77 +/- 0.02 in NTG and 5.84 +/- 0.01 in TG myofilament bundles (P < 0.05). After protein kinase A-dependent phosphorylation, the pCa(50) was 5.71 +/- 0.01 in NTG and 5.77 +/- 0. 02 in TG myofilament bundles (P < 0.05). Our results indicate that mutant alpha-Tm(Asp175Asn) increases myofilament Ca(2+)-sensitivity, which results in decreased relaxation rate and blunted response to beta-adrenergic stimulation.

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

1. To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene-encoded ssTnI. 2. The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3. Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP-dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild-type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non-transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4. The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild-type myocytes. However, the half-time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re-lengthening time of the transgenic cells. 5. These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6. Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to beta-adrenergic receptor stimulation.

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of alpha-TM, the murine alpha-TM gene was disrupted by homologous recombination. Homozygous alpha-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for alpha-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle alpha-TM mRNA, with no compensatory increase in transcript levels by striated muscle beta-TM or TM-30 isoforms. Surprisingly, this reduction in alpha-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle alpha-TM protein are produced and integrated in the myofibril. Quantification of alpha-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of alpha-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous alpha-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression.

We have tested the hypothesis that thyroid state may influence both the flow of cellular Ca2+ and the myofilament response to Ca2+ by effects on intracellular pH (pHi) and Na+ (Nai+). Single cardiac myocytes isolated from hypothyroid, euthyroid and hyperthyroid animals were loaded with fura-2/AM (Cai2+ probe), BCECF/AM (pHi probe) or SBFI/AM (Nai+ probe). Compared with hypothyroid animals, myocytes isolated from hyperthyroid rat hearts demonstrated a significant: (1) increase in extent of shortening; (2) decrease in the time to peak contraction; (3) increase in the peak amplitude of the fura-2 fluorescence ratio; (4) decrease in pHi (DeltapHi=0. 19+/-0.05); and (5) increase in Nai+ (DeltaNai+=2.88+/-0.55 mM). We have also compared pHi in Langendorff perfused hypo- and hyperthyroid rat hearts using NMR. We have found that hyperthyroid hearts are 0.15+/-0.03 pH units more acidic than hypothyroid hearts. Analysis of mRNA levels demonstrated that hyperthyroidism increased expression of both the Na+/Ca2+ exchanger and Na+/H+ antiporter, and decreased expression of Na+ channel mRNAs. These changes appear partially responsible for the observed changes in Nai+ and pHi. Our results provide the first evidence that changes in cardiac contractility associated with altered thyroid state not only involve effects on Ca2+, but may also involve changes in the response of the myofilaments to Cai2+mediated by altered pHi and Nai+., (Copyright 1997 Academic Press Limited.)

Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.

We measured the effects of the benzodiazocine derivative, CGP-48506 (5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1, 5-benzodiazocine-2,4-dione), on contraction of intact myocytes and permeabilized fibers of rat ventricular muscle. CGP-48506 is unique in that it is able to sensitize cardiac myofilaments to Ca2+, but unlike all other agents in this class, it is not an inhibitor of type III phosphodiesterase. When added to isolated intact myocytes, CGP-48506 significantly increased the amplitude of cell shortening with little or no change in the Ca2+ transient, as determined by the fluorescence ratio of fura 2. The late phase of the relation between fura 2 ratio and cell length was shifted to the left in the presence of CGP-48506. CGP-48506 also induced a relatively small decrease in diastolic length. However, compared with the thiadiazinone EMD-57033, CGP-48506 had a much smaller effect on diastolic length at concentrations in which there was a bigger inotropic effect. When added to solutions bathing detergent-extracted (skinned) fiber bundles, CGP-48506 increased maximum force. CGP-48506 also increased submaximal force and shifted the pGa-force relation to the left. However, compared with EMD-57033, there was less of an effect of CGP-48506 on force at relatively high pCa values. CGP-48506 did not alter Ca2+ binding to myofilament troponin C. CGP-48506 was able to reverse inhibition of contraction induced by butanedione monoxime both in intact cells and in skinned fiber bundles. Our results indicate that CGP-48506, like EMD-57033, is a positive inotropic agent working through a direct effect downstream from troponin C. CGP-48506, however, appears to have a unique mechanism resulting in less effect on diastolic function.

The effect of Thapsigargin (TG) shown to block selectively the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) on the SR Ca2+ content and contractions of single rat cadiomyocytes was investigated. In 80% of cells 10(-6) M TG blocked Ca2+ uptake by SR at rest, depleted the SR Ca2+, inhibited postrest potentiation and changed the pattern of response to premature contractions. Amplitude of steady state contractions was reduced to 11 +/- 4% of pre-TG control. In 20% of cells TG had similar effect on the SR function, however, amplitude of contractions was reduced to only 50 +/- 7% of control. It is concluded that the results obtained in 80% of the cells are compatible with the wide-spread notion that contractions of rat cardiac myocytes are activated predominantly by Ca2+ released from SR. However, population of myocytes of ventricular myocardium of this species is not homogeneous in this respect. In 20% of them contractions are activated by both sarcolemmal and SR Ca2+ contribution being approximately equal. These cells occupy intermediate position between guinea-pig cells and the bulk of rat cells.

Contractures initiated by 1 s superfusion of single myocytes of guinea-pig heart with 10 mM caffeine were used as a relative index of the SR Ca2+ content. Thapsigargin (Tg) in concentration 2 x 10(-7) M completely blocked Ca2+ uptake during electrical stimulation by the SR from which Ca2+ has been previously depleted by caffeine. Tg did not affect the SR Ca2+ content in the resting myocytes and did not block release of the SR Ca2+ during electrically stimulated contractions (ESCs). It is concluded that in guinea-pig myocytes Tg affects SR Ca2+ by selective blocking the SR Ca2+ uptake. The amplitude of steady state ESCs dropped to 68 +/- 5.4% (S.D., n = 20) of that of the pre-Tg control. Time to peak contraction increased from 454 +/- 82.4 ms to 820 +/- 157.4 ms and time of relaxation increased from 368 +/- 90.8 ms to 474 +/- 87 ms after the SR Ca2+ has been depleted by Tg. Rest decay of contractions, post-extrasystolic potentiation and post-rest potentiation were inhibited.

Inhibition of the Na-Ca exchange at the beginning of rest in isolated myocytes of the guinea-pig heart by means of superfusion with Na,Ca-free solution or 5.0 mM Ni2+ resulted in appearance of multiple phasic contractures. Contractures could not be initiated when the sarcoplasmic reticulum (SR) Ca2+ had been depleted by short (1 s) or steady state exposure to 10 mM caffeine, 0.1 microM ryanodine or due to rapid spontaneous release of the SR Ca2+ occurring sometimes at the beginning of rest. Superfusion with 2 x 10(-7) M thapsigargin, which blocked the SR Ca2+ uptake, prevented contractures otherwise initiated by superfusion with the Na,Ca-free solution. The frequency of spontaneous contractures was positively related to the rate of stimulation before rest and negatively related to the duration of rest before superfusion with the Na,Ca-free solution. It is proposed that in guinea-pig myocardium Ca2+ taken up by the SR from sarcoplasm or other cellular compartments like mitochondria, is released during diastole and rest to the subsarcolemmal space from which it is extruded by means of Na-Ca exchange. The release is a primary event not dependent on decrease of the resting sarcoplasmic free [Ca2+] by the outward Ca2+ transport. Inhibition of the Na-Ca exchange at the beginning of rest did not initiate any contractile response in rat myocytes. If the spontaneous contractures were already present, they were inhibited by superfusion with the Na,Ca-free solution. The result reflects the basic difference in the properties of SR of guinea-pig and rat.

The method of rapid superfusion of the single isolated ventricular myocytes of guinea-pig heart was used in order to inhibit the Na-Ca exchange throughout the physiological contraction-relaxation cycle. Superfusion of the cell at selected intervals during the contraction with the Na,Ca-free solution resulted in increase in its amplitude, increase in time to peak shortening and in delay of relaxation, albeit the cells relaxed before reperfusion of normal Tyrode solution. The largest increase in amplitude of contraction (to 134 +/- 16%) was observed when the effective exchange of the cell's environment was attained approximately 50 ms after the pulse stimulating contraction. The effects declined promptly when the delay was increased beyond 100 ms. In the cells treated with 10 mM caffeine superfusion with the Na,Ca-free solution after the delay of 50-100 ms resulted in decrease in extent of shortening. Increase in delay resulted in slight increase in extent of shortening with respect to control and strong inhibition of relaxation. The strongest effects were observed when the delay was approximately 200 ms. Superfusion of the normal cells and of the cells treated with caffeine between contractions resulted in slight potentiation of the next beat. It is concluded that Na-Ca exchange provides an important mechanism of relaxation and outward Ca2+ transport in the physiological contraction of the ventricular cardiomyocyte.

We investigated the effect of 2-methyl-1,4-naphtoquinone (Menadione) on sarcoplasmic reticulum (SR) Ca2+ content and electrically stimulated contractions (ESCs) of single isolated myocytes of guinea-pig ventricular myocardium. The contractures initiated by means of microinjections of caffeine into the close vicinity of the cell were used as an indirect index of the SR Ca2+ content. Superfusion of the cells for 45 min with Menadione resulted in gradual disappearance of contractile responses to caffeine, prolongation of time to peak amplitude of ESCs by 48 +/- 15% and complete inhibition of postrest and postextrasystolic potentiation. These results are consistent with those of Floreani and Carpenedo (7) who found that Menadione strongly inhibits the SR Ca2+ ATPase. Despite depletion of the SR Ca2+ the amplitude of ESCs did not change which suggests that contractions were initiated in the cells treated with Menadione by Ca2+ derived from the sources other than the SR.