Evidence for heterogeneous subsarcolemmal Na+ levels in rat ventricular myocytes.

The intracellular Na+ concentration ([Na+]) regulates cardiac contractility. Previous studies have suggested that subsarcolemmal [Na+] is higher than cytosolic [Na+] in cardiac myocytes, but this concept remains controversial. Here, we used electrophysiological experiments and mathematical modeling to test whether there are subsarcolemmal pools with different [Na+] and dynamics compared with the bulk cytosol in rat ventricular myocytes. A Na+ dependency curve for Na+-K+-ATPase (NKA) current was recorded with symmetrical Na+ solutions, i.e., the same [Na+] in the superfusate and internal solution. This curve was used to estimate [Na+] sensed by NKA in other experiments. Three experimental observations suggested that [Na+] is higher near NKA than in the bulk cytosol: 1) when extracellular [Na+] was high, [Na+] sensed by NKA was ~6 mM higher than the internal solution in quiescent cells; 2) long trains of Na+ channel activation almost doubled this gradient; compared with an even intracellular distribution of Na+, the increase of [Na+] sensed by NKA was 10 times higher than expected, suggesting a local Na+ domain; and 3) accumulation of Na+ near NKA after trains of Na+ channel activation dissipated very slowly. Finally, mathematical models assuming heterogeneity of [Na+] between NKA and the Na+ channel better reproduced experimental data than the homogeneous model. In conclusion, our data suggest that NKA-sensed [Na+] is higher than [Na+] in the bulk cytosol and that there are differential Na+ pools in the subsarcolemmal space, which could be important for cardiac contractility and arrhythmogenesis. NEW & NOTEWORTHY Our data suggest that the Na+-K+-ATPase-sensed Na+ concentration is higher than the Na+ concentration in the bulk cytosol and that there are differential Na+ pools in the subsarcolemmal space, which could be important for cardiac contractility and arrhythmogenesis. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/heterogeneous-sodium-in-ventricular-myocytes/ .

Hypokalaemia induces Ca²âº overload and Ca²âº waves in ventricular myocytes by reducing Na⁺,K⁺-ATPase α2 activity.

KEY POINTS: Hypokalaemia is a risk factor for development of ventricular arrhythmias. In rat ventricular myocytes, low extracellular K(+) (corresponding to clinical moderate hypokalaemia) increased Ca(2+) wave probability, Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load and induced SR Ca(2+) leak. Low extracellular K(+) reduced Na(+),K(+)-ATPase (NKA) activity and hyperpolarized the resting membrane potential in ventricular myocytes. Both experimental data and modelling indicate that reduced NKA activity and subsequent Na(+) accumulation sensed by the Na(+), Ca(2+) exchanger (NCX) lead to increased Ca(2+) transient amplitude despite concomitant hyperpolarization of the resting membrane potential. Low extracellular K(+) induced Ca(2+) overload by lowering NKA α2 activity. Triggered ventricular arrhythmias in patients with hypokalaemia may therefore be attributed to reduced NCX forward mode activity linked to an effect on the NKA α2 isoform. ABSTRACT: Hypokalaemia is a risk factor for development of ventricular arrhythmias. The aim of this study was to determine the cellular mechanisms leading to triggering of arrhythmias in ventricular myocytes exposed to low Ko. Low Ko, corresponding to moderate hypokalaemia, increased Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load, SR Ca(2+) leak and Ca(2+) wave probability in field stimulated rat ventricular myocytes. The mechanisms leading to Ca(2+) overload were examined. Low Ko reduced Na(+),K(+)-ATPase (NKA) currents, increased cytosolic Na(+) concentration and increased the Na(+) level sensed by the Na(+), Ca(2+) exchanger (NCX). Low Ko also hyperpolarized the resting membrane potential (RMP) without significant alterations in action potential duration. Experiments in voltage clamped and field stimulated ventricular myocytes, along with mathematical modelling, suggested that low Ko increases the Ca(2+) transient amplitude by reducing NKA activity despite hyperpolarization of the RMP. Selective inhibition of the NKA α2 isoform by low dose ouabain abolished the ability of low Ko to reduce NKA currents, to increase Na(+) levels sensed by NCX and to increase the Ca(2+) transient amplitude. We conclude that low Ko, within the range of moderate hypokalaemia, increases Ca(2+) levels in ventricular myocytes by reducing the pumping rate of the NKA α2 isoform with subsequent Na(+) accumulation sensed by the NCX. These data highlight reduced NKA α2 -mediated control of NCX activity as a possible mechanism underlying triggered ventricular arrhythmias in patients with hypokalaemia.

Impaired left ventricular mechanical and energetic function in mice after cardiomyocyte-specific excision of Serca2.

Sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA)2 transports Ca2+ from the cytosol into the sarcoplasmic reticulum of cardiomyocytes and is essential for maintaining myocardial Ca2+ handling and thus the mechanical function of the heart. SERCA2 is a major ATP consumer in excitation-contraction coupling but is regarded to contribute to energetically efficient Ca2+ handling in the cardiomyocyte. Previous studies using cardiomyocyte-specific SERCA2 knockout (KO) mice have demonstrated that decreased SERCA2 activity reduces the Ca2+ transient amplitude and induces compensatory Ca2+ transport mechanisms that may lead to more inefficient Ca2+ transport. In this study, we examined the relationship between left ventricular (LV) function and myocardial O2 consumption (MVo2) in ex vivo hearts from SERCA2 KO mice to directly measure how SERCA2 elimination influences mechanical and energetic features of the heart. Ex vivo hearts from SERCA2 KO hearts developed mechanical dysfunction at 4 wk and demonstrated virtually no working capacity at 7 wk. In accordance with the reported reduction in Ca2+ transient amplitude in cardiomyocytes from SERCA2 KO mice, work-independent MVo2 was decreased due to a reduced energy cost of excitation-contraction coupling. As these hearts also showed a marked impairment in the efficiency of chemomechanical energy transduction (contractile efficiency, i.e, work-dependent MVo2), hearts from SERCA2 KO mice were found to be mechanically inefficient. This ex vivo evaluation of mechanical and energetic function in hearts from SERCA2 KO mice brings together findings from previous experimental and mathematical modeling-based studies and demonstrates that reduced SERCA2 activity not only leads to mechanical dysfunction but also to energetic dysfunction.

Cardiac sodium transport and excitation-contraction coupling.

The excitation-contraction coupling (EC-coupling) links membrane depolarization with contraction in cardiomyocytes. Ca(2+) induced opening of ryanodine receptors (RyRs) leads to Ca(2+) induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) into the dyadic cleft between the t-tubules and SR. Ca(2+) is removed from the cytosol by the SR Ca(2+) ATPase (SERCA2) and the Na,Ca-exchanger (NCX). The NCX connects cardiac Ca(2+) and Na(+)-transport, leading to Na(+)-dependent regulation of EC-coupling by several mechanisms of which some still lack firm experimental evidence. Firstly, NCX might contribute to CICR during an action potential (AP) as Na(+)-accumulation at the intracellular site together with depolarization will trigger reverse mode exchange bringing Ca(2+) into the dyadic cleft. The controversial issue is the nature of the compartment in which Na(+) accumulates. It seems not to be the bulk cytosol, but is it part of a widespread subsarcolemmal space, a localized microdomain ("fuzzy space"), or as we propose, a more localized "spot" to which only a few membrane proteins have shared access (nanodomains)? Also, there seems to be spots where the Na,K-pump (NKA) will cause local Na(+) depletion. Secondly, Na(+) determines the rate of cytosolic Ca(2+) removal and SR Ca(2+) load by regulating the SERCA2/NCX-balance during the decay of the Ca(2+) transient. The aim of this review is to describe available data and current concepts of Na(+)-mediated regulation of cardiac EC-coupling, with special focus on subcellular microdomains and the potential roles of Na(+) transport proteins in regulating CICR and Ca(2+) extrusion in cardiomyocytes. We propose that voltage gated Na(+) channels, NCX and the NKA α2-isoform all regulate cardiac EC-coupling through control of the "Na(+) concentration in specific subcellular nanodomains in cardiomyocytes. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes."

Intrinsic skeletal muscle alterations in chronic heart failure patients: a disease-specific myopathy or a result of deconditioning?

Chronic heart failure (CHF) patients frequently experience impaired exercise tolerance due to skeletal muscle fatigue. Studies suggest that this in part is due to intrinsic alterations in skeletal muscle of CHF patients, often interpreted as a disease-specific myopathy. Knowledge about the mechanisms underlying these skeletal muscle alterations is of importance for the pathophysiological understanding of CHF, therapeutic approach and rehabilitation strategies. We here critically review the evidence for skeletal muscle alterations in CHF, the underlying mechanisms of such alterations and how skeletal muscle responds to training in this patient group. Skeletal muscle characteristics in CHF patients are very similar to what is reported in response to chronic obstructive pulmonary disease (COPD), detraining and deconditioning. Furthermore, skeletal muscle alterations observed in CHF patients are reversible by training, and skeletal muscle of CHF patients seems to be at least as trainable as that of matched controls. We argue that deconditioning is a major contributor to the skeletal muscle dysfunction in CHF patients and that further research is needed to determine whether, and to what extent, the intrinsic skeletal muscle alterations in CHF represent an integral part of the pathophysiology in this disease.

Preserved metabolic reserve capacity in skeletal muscle of post-infarction heart failure patients.

It has been proposed that exercise capacity during whole body exercise in post-infarction congestive heart failure (CHF) patients is limited by skeletal muscle function. We therefore investigated the balance between cardiopulmonary and muscular metabolic capacity. CHF patients (n=8) and healthy subjects (HS, n=12) were included. Patients with coronary artery disease (CAD, n=8) were included as a control for medication. All subjects performed a stepwise incremental load test during bicycling (∼24 kg muscle mass), two-legged knee extensor (2-KE) exercise (∼4 kg muscle mass) and one-legged knee extensor (1-KE) exercise (∼2 kg muscle mass). Peak power and peak pulmonary oxygen uptake (VO(2peak) ) increased and muscle-specific VO(2peak) decreased with an increasing muscle mass involved in the exercise. Peak power and VO(2peak) were lower for CHF patients than HS, with values for CAD patients falling between CHF patients and HS. During bicycling, all groups utilized 24-29% of the muscle-specific VO(2peak) as measured during 1-KE exercise, with no difference between the groups. Hence, the muscle metabolic reserve capacity during whole body exercise is not different between CHF patients and HS, indicating that appropriately medicated and stable post-infarction CHF patients are not more limited by intrinsic skeletal muscle properties during whole body exercise than HS.

Calcium dynamics in the ventricular myocytes of SERCA2 knockout mice: A modeling study.

We describe a simulation study of Ca²(+) dynamics in mice with cardiomyocyte-specific conditional excision of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) gene, using an experimental data-driven biophysically-based modeling framework. Previously, we reported a moderately impaired heart function measured in mice at 4 weeks after SERCA2 gene deletion (knockout (KO)), along with a >95% reduction in the level of SERCA2 protein. We also reported enhanced Ca²(+) flux through the L-type Ca²(+) channels and the Na(+)/Ca²(+) exchanger in ventricular myocytes isolated from these mice, compared to the control Serca2(flox/flox) mice (flox-flox (FF)). In the current study, a mathematical model-based analysis was applied to enable further quantitative investigation into changes in the Ca²(+) handling mechanisms in these KO cardiomyocytes. Model parameterization based on a wide range of experimental measurements showed a 67% reduction in SERCA activity and an over threefold increase in the activity of the Na(+)/Ca²(+) exchanger. The FF and KO models were then validated against experimentally measured [Ca²(+)](i) transients and experimentally estimated sarco(endo)plasmic reticulum (SR) function. Simulation results were in quantitative agreement with experimental measurements, confirming that sustained [Ca²(+)](i) transients could be maintained in the KO cardiomyocytes despite severely impaired SERCA function. In silico analysis shows that diastolic [Ca²(+)](i) rises sharply with progressive reductions in SERCA activity at physiologically relevant pacing frequencies. Furthermore, an analysis of the roles of the compensatory mechanisms revealed that the major combined effect of the compensatory mechanisms is to lower diastolic [Ca²(+)](i). Finally, by using a comprehensive sensitivity analysis of the role of all cellular calcium handling mechanisms, we show that the combination of upregulation of the Na(+)/Ca²(+) exchanger and increased L-type Ca²(+) current is the most effective means to maintain diastolic and systolic calcium levels after loss of SERCA function.

Effects of treatment with a 5-HT4 receptor antagonist in heart failure.

BACKGROUND AND PURPOSE: Positive inotropic responses (PIR) to 5-hydroxytryptamine (5-HT) are induced in the left ventricle (LV) in rats with congestive heart failure (CHF); this is associated with upregulation of the G(s)-coupled 5-HT(4) receptor. We investigated whether chronic 5-HT(4) receptor blockade improved cardiac function in CHF rats. EXPERIMENTAL APPROACH: Rats were given either the 5-HT(4) antagonist SB207266 (0.5 mg kg(-1) 24h(-1); MI(int)) or placebo (MI(pl)) through mini-osmotic pumps for 6 weeks subsequent to induction of post-infarction CHF. In vivo cardiac function and ex vivo responses to isoprenaline or 5-HT were evaluated using echocardiography and isolated LV papillary muscles, respectively. mRNA levels were investigated using real-time quantitative RT-PCR. KEY RESULTS: LV diastolic function improved, with 4.6% lower LV diastolic diameter and 24.2% lower mitral flow deceleration in MI(int) compared to MI(pl). SB207266 reduced LV systolic diameter by 6.1%, heart weight by 10.2% and lung weight by 13.1%. The changes in posterior wall thickening and shortening velocity, cardiac output, LV systolic pressure and (dP/dt)(max), parameters of LV systolic function, did not reach statistical significance. The PIR to isoprenaline (10 microM) increased by 36% and the response to 5-HT (10 microM) decreased by 57% in MI(int) compared to MI(pl). mRNA levels for ANP, 5-HT(4(b)) and 5-HT(2A) receptors, MHCbeta, and the MHCbeta/MHCalpha -ratio were not significantly changed in MI(int) compared to MI(pl). CONCLUSIONS AND IMPLICATIONS: Treatment with SB207266 to some extent improved in vivo cardiac function and ex vivo myocardial function, suggesting a possible beneficial effect of treatment with a 5-HT(4) receptor antagonist in CHF.

Cross-reinnervation changes the expression patterns of the monocarboxylate transporters 1 and 4: An experimental study in slow and fast rat skeletal muscle.

The monocarboxylate transporters 1 and 4 are expressed in brain as well as in skeletal muscle and play important roles in the energy metabolism of both tissues. In brain, monocarboxylate transporter 1 occurs in astrocytes, ependymocytes, and endothelial cells while monocarboxylate transporter 4 appears to be restricted to astrocytes. In muscle, monocarboxylate transporter 1 is enriched in oxidative muscle fibers whereas monocarboxylate transporter 4 is expressed in all fibers, with the lowest levels in oxidative fiber types. The mechanisms regulating monocarboxylate transporter 1 and monocarboxylate transporter 4 expression are not known. We hypothesized that the expression of these transporters would be sensitive to long term changes in metabolic activity level. This hypothesis can be tested in rat skeletal muscle, where permanent changes in activity level can be induced by cross-reinnervation. We transplanted motor axons originally innervating the fast-twitch extensor digitorum longus muscle to the slow-twitch soleus muscle and vice versa. Four months later, microscopic analysis revealed transformation of muscle fiber types in the cross-reinnervated muscles. Western blot analysis showed that monocarboxylate transporter 1 was increased by 140% in extensor digitorum longus muscle and decreased by 30% in soleus muscle after cross-reinnervation. In contrast, cross-reinnervation induced a 62% decrease of monocarboxylate transporter 4 in extensor digitorum longus muscle and a 1300% increase in soleus muscle. Our findings show that cross-reinnervation causes pronounced changes in the expression levels of monocarboxylate transporter 1 and monocarboxylate transporter 4, probably as a direct consequence of the new pattern of nerve impulses. The data indicate that the mode of innervation dictates the expression of monocarboxylate transporter proteins in the target cells and that the change in monocarboxylate transporter isoform profile is an integral part of the muscle fiber transformation that occurs after cross-reinnervation. Our findings support the hypothesis that the expression of monocarboxylate transporter 1 and monocarboxylate transporter 4 in excitable tissues is regulated by activity.

Temperature-dependent skeletal muscle dysfunction in rats with congestive heart failure.

Abnormalities in the excitation-contraction coupling of slow-twitch muscle seem to explain the slowing and increased fatigue observed in congestive heart failure (CHF). However, it is not known which elements of the excitation-contraction coupling might be affected. We hypothesize that the temperature sensitivity of contractile properties of the soleus muscle might be altered in CHF possibly because of alterations of the temperature sensitivity of intracellular Ca(2+) handling. We electrically stimulated the in situ soleus muscle of anesthetised rats that had 6-wk postinfarction CHF using 1 and 50 Hz and using a fatigue protocol (5-Hz stimulation for 30 min) at 35, 37, and 40 degrees C. Ca(2+) uptake and release were measured in sarcoplasmic reticulum vesicles at various temperatures. Contraction and relaxation rates of the soleus muscle were slower in CHF than in sham at 35 degrees C, but the difference was almost absent at 40 degrees C. The fatigue protocol revealed that force development was more temperature sensitive in CHF, whereas contraction and relaxation rates were less temperature sensitive in CHF than in sham. The Ca(2+) uptake and release rates did not correlate to the difference between CHF and sham regarding contractile properties or temperature sensitivity. In conclusion, the discrepant results regarding altered temperature sensitivity of contraction and relaxation rates in the soleus muscle of CHF rats compared with Ca(2+) release and uptake rates in vesicles indicate that the molecular cause of slow-twitch muscle dysfunction in CHF is not linked to the intracellular Ca(2+) cycling.

Defective excitation-contraction coupling in hearts of rats with congestive heart failure.

AIM: We examined the cellular basis for depressed cardiac contractility in rats with congestive heart failure (CHF) secondary to myocardial infarction. METHODS: Six weeks after ligation of the left coronary artery, CHF was confirmed by haemodynamic measures and echocardiographic demonstration of reduced myocardial contractility in vivo. Papillary muscles from CHF animals developed less force than those from sham operated (SHAM) animals. Cell shortening was measured in isolated ventricular myocytes voltage-clamped with high resistance electrodes. Ca2+ transients were measured in fluo-4 loaded myocytes. RESULTS: Contractions triggered by depolarizing test steps from a post conditioning potential of -70 mV were significantly smaller and had significantly reduced velocity of shortening in CHF compared with SHAM myocytes. However, contractions initiated from -40 mV, were similar in amplitude and velocity of shortening in CHF and SHAM cells. L-type Ca2+ current was not significantly different between CHF and SHAM cells, whether activated from -70 or -40 mV. Therefore, in SHAM cells, excitation-contraction coupling exhibited higher gain when contractions were initiated from negative (-70 mV), as compared with depolarized potentials (-40 mV). However, in CHF myocytes, excitation-contraction coupling gain was selectively depressed with steps from -70 mV. This depression of gain in CHF was not accompanied by a significant reduction in sarcoplasmic reticulum Ca2+ content. Isoproterenol increased Ca2+ transients less in CHF than SHAM myocytes. CONCLUSION: In this post-infarction model of CHF, the contractile deficit was voltage dependent and the gain of excitation-contraction coupling was selectively depressed for contractions initiated negative to -40 mV.

Muscle dysfunction during exercise of a single skeletal muscle in rats with congestive heart failure is not associated with reduced muscle blood supply.

AIM: Inadequate muscle blood flow is a possible explanation for reduced fatigue resistance in patients with congestive heart failure (CHF). METHODS: In rats with post-infarction CHF we electrically stimulated the soleus muscle (SOL) in situ with intact blood supply. Contractile properties, blood flow, high-energy phosphates and metabolites were measured during 30 min of intermittent stimulation, and in addition capillarization of SOL was recorded. RESULTS: During stimulation, SOL contracted more slowly in rats with CHF compared with sham-operated rats. However, the blood flow in SOL was unaltered and capillary density was maintained in CHF rats. Further, the content of ATP, ADP, AMP, NAD, CrP, P(i) and lactate in SOL was not different between the groups. CONCLUSION: The cause of contractile dysfunction in a single exercising skeletal muscle in rats with CHF cannot be explained simply by reduced blood supply. In addition, absence of changes in high-energy phosphates and metabolites indicate that the oxidative metabolism of SOL is intact in rats with CHF.

Analysis of energy metabolism and mechanism of loop diuretics in the thick ascending limb of Henle's loop in dog kidneys.

AIM: The thick ascending limb of Henle's loop (TALH) absorbs up to 40% of filtered NaCl in volume-expanded dogs. To examine if a fraction of this absorption is passive, NaHCO3 absorption and associated NaCl absorption in proximal tubules were inhibited by acetazolamide, a carbonic anhydrase inhibitor. RESULTS: Ouabain, a specific inhibitor of Na,K-ATPase activity, reduced the remaining NaCl absorption and renal oxygen consumption in a ratio DeltaNa/DeltaO2 = 18, as expected for active transport. However, the responses to two loop diuretics were DeltaNa/DeltaO2 = 24 for ethacrynic acid and DeltaNa/DeltaO2 = 30 for bumetanide. Both loop diuretics induced potassium secretion. By superimposing ouabain potassium secretion was stopped and DeltaNa/DeltaO2 = 18 restored. Replacement of half of the circulating NaCl with Na2SO4 gave stop-flow pattern similar to those obtained after ethacrynic acid. CONCLUSIONS: Low entry of some sodium ions thorugh the apical membrane is permitted despite low chloride supply or blockade by loop diuretics of chloride entry by the Na-K-2Cl transporter. Continued Na-K-ATPase activity causes secretion of potassium ions through the apical ion channel, ethacrynic acid being more kaliuretic and less natriuretic than bumetanide. Greater paracellular recycling of sodium ions after bumetanide maintains ionic balance. In contrast, under normal conditions excess entry of chloride by the Na-K-2Cl-transporter leads to paracellular back-diffusion of chloride rather than paracellular absorption of sodium ions, consistent with DeltaNa/DeltaO2 = 18 after ouabain. Thus all NaCl transport along TALH is active in vivo, whereas absorption of other cations, such as lithium, probably is passive.

Increased contribution of alpha 1- vs. beta-adrenoceptor-mediated inotropic response in rats with congestive heart failure.

AIM: In failing myocardium the mechanical response to beta-adrenoceptor stimulation is attenuated. Alternative signalling systems might provide inotropic support when the beta-adrenoceptor system is dysfunctioning. Accordingly, the inotropic responses to alpha 1- and beta-adrenoceptor stimulation by the endogenous adrenoceptor agonist noradrenaline in non-failing and failing rat hearts were compared. METHODS: Chronic heart failure was induced in male Wistar rats by coronary artery ligation. Corresponding sham groups were prepared. After 6 weeks, papillary muscles from non-failing and failing hearts were isolated. Receptor binding studies were performed in the corresponding myocardium. The alpha 1-adrenoceptor-mediated inotropic response was not changed while the beta-adrenoceptor-mediated response was substantially reduced in failing compared with non-failing myocardium. RESULTS: No change in potency for the agonists was observed at the alpha 1-adrenoceptors, while an increased potency for the agonists at the beta-adrenoceptors was found during heart failure. The lusitropic response to beta-adrenoceptor stimulation was intact during heart failure. No over all change in affinity or number of either adrenoceptor type was observed in receptor binding studies. The alpha 1-adrenoceptor-mediated inotropic response became dominating compared with the beta-adrenoceptor-mediated one in failing rat myocardium in contrast to the dominating role of the latter in non-failing myocardium. The attenuation of the beta-adrenoceptor-mediated inotropic response in rat failing myocardium was not because of a reduced number of receptors. CONCLUSION: Increasing contractility through stimulation of alpha 1-adrenoceptors in situ by the endogenous agonist may be an alternative way of inotropic support during heart failure and even more so during beta-adrenoceptor blockade.

Reduced calcium tolerance in rat cardiomyocytes after myocardial infarction.

During ischaemia and reperfusion the intracellular Na+ concentration is elevated in the cardiomyocytes and the cells are depolarized, both favouring reverse mode Na,Ca-exchange loading of the cell with Ca2+. We examined whether cardiomyocytes from rats with congestive heart failure (CHF) and younger rats (HINCX) which both have a high expression of the Na,Ca-exchanger protein (NCX) showed reduced tolerance to extracellular Ca2+. The CHF was induced in Isofluran anaesthetized rats by left coronary artery ligation. Isolated cardiomyocytes were loaded with Fura-2AM and 140 mm Na+ and exposed to 0.05 mm Ca2+. Expression of the Na,Ca-exchanger protein was analysed. Fura-2 340/380 ratio rose more rapidly in HINCX and CHF than in SHAM, and the rise was abolished by Ni2+. Hypercontracture developed more frequently in HINCX and CHF than in SHAM cells. The amount of NCX was 54% higher in HINCX and 76% higher in CHF compared with SHAM. Na+-loaded cardiomyocytes from CHF and HINCX rats are more susceptible to Ca2+ overload than SHAM cells because of the increased capacity for Na,Ca-exchange.

Surgical manipulation, but not moderate exercise, is associated with increased cytokine mRNA expression in the rat soleus muscle.

Interleukin (IL)-6 production in contracting skeletal muscle and IL-6 concentration in plasma are increased after prolonged and strenuous exercise. However, as tissue stress or damage are unspecific triggers of increased cytokine levels, we examined whether moderate muscle activity is an independent stimulus for cytokine expression, and to which extent invasive procedures might affect the results. Soleus muscles were isolated from sedentary rats or from rats that had been running on a treadmill at moderate intensity (70% of maximal oxygen uptake) for 1 h. In another group the soleus muscle was prepared in situ and stimulated intermittently at 5 Hz for 1 h, so that maximal developed force declined by 30%. In situ prepared soleus muscles not subjected to electrical stimulation were used as controls. Messenger RNA (mRNA) expression of 11 cytokines was analysed in the soleus muscles using multiprobe RNAse protection assay, and IL-6 plasma concentration was measured by enzyme-linked immunosorbent assay. Treadmill exercise did not affect the mRNA expression of any of the measured cytokines in the soleus muscle. Irrespective of electrical stimulation, mRNA expression of IL-6 and IL-1beta were significantly increased in the surgically manipulated soleus muscles. Interleukin-6 plasma concentration was not affected by treadmill running or electrical stimulation. Conclusion, gentle surgical manipulation is a strong stimulus for IL-6 and IL-1beta mRNA synthesis in skeletal muscle, whereas exercise or electrical muscle stimulation at moderate intensity does not independently affect cytokine mRNA levels in the contracting soleus.

Muscle contractile properties during intermittent nontetanic stimulation in rat skeletal muscle.

To examine changes in contractile properties and mechanisms of fatigue during submaximal nontetanic skeletal muscle activity, in situ perfused soleus (60-min protocol) and extensor digitorum longus (EDL; 10-min protocol) muscles of the rat were electrically stimulated intermittently at low frequency. The partly fused trains of contractions showed a two-phase change in appearance. During the first phase, relaxation slowed, one-half relaxation time increased, and maximal relaxation first derivative of force (dF/dt) decreased. Developed force during the trains was reduced and was closely related to the rate of relaxation in this first phase. During the second phase, relaxation became faster again, one-half relaxation time decreased, and force returned to resting levels between contractions in a train. In contrast, developed force remained reduced, so that peak force of the contractions was 51% (soleus) and 30% (EDL) of control. In the soleus muscle, the changes in contractile properties were not related to ATP, creatine phosphate, or lactate content. The changes in contractile properties fit best with a mechanism of fatigue involving changes in Ca(2+) handling by the sarcoplasmic reticulum.

Upregulation of the cardiac monocarboxylate transporter MCT1 in a rat model of congestive heart failure.

BACKGROUND: Cardiac metabolism becomes more dependent on carbohydrates in congestive heart failure (CHF), and lactate may be used as an important respiratory substrate. Monocarboxylate transporter 1 (MCT1) promotes cotransport of lactate and protons into and out of heart cells and conceivably flux of lactate between cells, because it is abundantly present in the intercalated disk. METHODS AND RESULTS: Six weeks after induction of myocardial infarction (MI) in Wistar rats, left ventricular end-diastolic pressures were >15 mm Hg, signifying CHF. MCT1 and connexin43 protein levels in CHF were 260% and 20%, respectively, of those in sham-operated animals (Sham), and the corresponding mRNA signals were 181% and not significantly changed, respectively. Confocal laserscan immunohistochemistry and quantitative immunogold cytochemistry showed that MCT1 density was much higher in CHF than in Sham both at the surface membrane and in the intercalated disk. In CHF, a novel intracellular pool of MCT1 appeared to be associated with cisternae, some close to the T tubules. In contrast, connexin43 particles, seen exclusively at gap junctions, were substantially fewer. Maximum lactate uptake was 107+/-15 mmol. L(-1). min(-1) in CHF and 42+/-6 mmol. L(-1). min(-1) in Sham cells (P<0.05). The K(m) values were between 7 and 9 mmol/L (P=NS). CONCLUSIONS: In cardiomyocytes from CHF rats, (1) the amount of functional MCT1 in the sarcolemma, including in the intercalated disk, is increased several-fold; (2) a new intracellular pool of MCT1 appears; (3) another disk protein, connexin43, is much reduced; and (4) increased reliance on lactate and other monocarboxylates (eg, pyruvate) could provide tight metabolic control of high-energy phosphates.

Monocyte chemoattractant protein-1 enhances and interleukin-10 suppresses the production of inflammatory cytokines in adult rat cardiomyocytes.

OBJECTIVE: Chemokines control the migration of leukocytes to inflamed tissue, and in particular monocyte chemoattractant protein (MCP)-1 has been implicated in the pathogenesis of several cardiovascular disorders such as chronic heart failure (CHF) and myocarditis. We hypothesised that MCP-1 may directly contribute to an inflammatory response in the cardiomyocytes, and in the present study we examined in adult rat cardiomyocytes: (i) the effect of tumour necrosis factor (TNF)alpha on MCP-1 production, (ii) the effect of MCP-1 on production of other inflammatory cytokines, and (iii) if the anti-inflammatory cytokine interleukin (IL)-10 could suppress any TNFalpha-induced MCP- 1 production. METHODS: We used enzyme immunoassays, RNase protection assays and slot blot analysis to measure protein and mRNA levels of various cytokines in adult rat cardiomyocyte cultures. RESULTS: (i) We found a approximately 6.4-fold increase of the MCP-1 level accompanied by an increase in MCP-1 mRNA accumulation in cardiomyocyte cultures after TNFalpha stimulation. (ii) In contrast, TNFalpha had no effect on IL-10 and only a modest effect on IL-1beta and IL-6 levels in these cells. (iii) Importantly, MCP-1 stimulated inflammatory response in cardiomyocytes by enhancing IL- 1beta and IL-6 levels in these cells as found at both the protein and mRNA level. (iv) Co-stim-ulation with IL-10 resulted in a approximately 55% reduction in TNFalpha-stimulated MCP-1 levels in cardiomyocyte culture supernatants. CONCLUSION: The present study demonstrates for the first time that MCP- 1 can directly affect cardiomyocytes, and we introduce MCP-1 as a potential enhancer and IL- 10 as a potential suppresser of inflammatory responses within the myocardium.

Skeletal muscle disorders in heart failure.

Heart failure is associated with reduction of exercise capacity that cannot be solely ascribed to reduced maximal oxygen uptake (VdotO2max). Therefore, research has focused on changes in skeletal muscle morphology, metabolism and function. Factors that can cause such changes in skeletal muscle comprise inactivity, malnutrition, constant or repeated episodes of inadequate oxygen delivery and prolonged exposure to altered neurohumoural stimuli. Most of these factors are not specific for the heart failure condition. On the other hand, heart failure is more than one clinical condition. Congestive heart failure (CHF) develops gradually as a result of deteriorating contractility of the viable myocardium, myocardial failure. Is it possible that development of this contractile deficit in the myocardium is paralleled by a corresponding contractile deficit of the skeletal muscles? This question cannot be answered today. Both patient studies and experimental studies support that there is a switch to a faster muscle phenotype and energy metabolism balance is more anaerobic. The muscle atrophy seen in many patients is not so evident in experimental studies. Few investigators have studied contractile function. Both fast twitch and slow twitch muscles seem to become slower, not faster as might be expected, and this is possibly linked to slower intracellular Ca2+ cycling. The neurohumoural stimuli that can cause this change are not known, but recently it has been reported that several cytokines are increased in CHF patients. Thus, the changes seen in skeletal muscles during CHF are partly secondary to inactivity, but the possibility remains that the contractility is altered because of intracellular changes of Ca2+ metabolism that are also seen in the myocardium.