Biomedical subjects

Paul M L Janssen

Publications and source records attributed to Paul M L Janssen.

At least 19 recordsLinked to original sources

PubMed2007

Frequency-dependent acceleration of relaxation involves decreased myofilament calcium sensitivity.

The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultrathin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura 2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic intracellular calcium concentration with increased frequency. Steady-state force-intracellular calcium concentration relationships were measured at frequencies 1, 2, 3, and 4 Hz at 37 degrees C using potassium-induced contractures. EC(50) significantly and gradually increased with frequency, from 475 +/- 64 nM at 1 Hz to 1,004 +/- 142 nM at 4 Hz (P < 0.05) and correlated with the corresponding changes in half relaxation time. No significant changes in maximal active force development or in the myofilament cooperativity coefficient were found. Myofilament protein phosphorylation was assessed using Pro-Q Diamond staining on protein gels of trabeculae frozen at either 1 or 4 Hz, revealing troponin I and myosin light chain-2 phosphorylation associated with the myofilament desensitization. We conclude that myofilament calcium sensitivity is substantially and significantly decreased at higher frequencies, playing a prominent role in frequency-dependent acceleration of relaxation.

Actin Cytoskeleton↗

PubMed2006

SERCA overexpression reduces hydroxyl radical injury in murine myocardium.

Hydroxyl radicals (*OH) are involved in the pathogenesis of ischemia-reperfusion injury and are observed in clinical situations, including acute heart failure, stroke, and myocardial infarction. Acute transient exposure to *OH causes an intracellular Ca(2+) overload and leads to impaired contractility. We investigated whether upregulation of sarcoplasmic reticulum Ca(2+)-ATPase function (SERCA) can attenuate *OH-induced dysfunction. Small, contracting right ventricular papillary muscles from wild-type (WT) SERCA1a-overexpressing (transgenic, TG) and SERCA2a heterogeneous knockout (HET) mice were directly exposed to *OH. This brief 2-min exposure led to a transient elevation of diastolic force (F(dia)) and depression of developed force (F(dev)). In WT mice, F(dia) increased to 485 +/- 49% and F(dev) decreased to 11 +/- 3%. In sharp contrast, in TG mice F(dia) increased only to 241 +/- 17%, whereas F(dev) decreased only to 51 +/- 5% (P < 0.05 vs. WT). In HET mice, F(dia) rose more than WT (to 597 +/- 20%, P < 0.05), whereas F(dev) was reduced in a similar amount. After approximately 45 min after *OH exposure, a new steady state was reached: F(dev) returned to 37 +/- 6% and 32 +/- 6%, whereas F(dia) came back to 238 +/- 28% and 292 +/- 17% in WT and HET mice, respectively. In contrast, the sustained dysfunction was significantly less in TG mice: F(dia) and F(dev) returned to 144 +/- 20% and 67 +/- 6%, respectively. Before exposure to *OH, there is decrease in phospholamban (PLB) phosphorylation at Ser16 (pPLBSer16) and PLB phosphorylation at Thr17 (pPLBThr17) in TG mice and an increase in pPLBSer16 and pPLBThr17 in HET mice versus WT. After exposure to *OH there is decrease in pPLBSer16 in WT, TG, and HET mice but no significant change in the level of pPLBThr17 in any group. The results indicate that SERCA overexpression can reduce the *OH-induced contractile dysfunction in murine myocardium, whereas a reduced SR Ca(2+)-ATPase activity aggravates this injury. Loss of pPLB levels at Ser16 likely amplifies the differences observed in injury response.

Animals↗

PubMed2006

LAMP-2 deficient mice show depressed cardiac contractile function without significant changes in calcium handling.

OBJECTIVE: Mutations in the highly glycosylated lysosome associated membrane protein-2 (LAMP-2) cause, as recently shown, familial Danon disease with mental retardation, mild myopathy and fatal cardiomyopathy. Extent and basis of the contractile dysfunction is not completely understood. METHODS: In LAMP-2 deficient mice, we investigated cardiac function in vivo using Doppler-echocardiography and contractile function in vitro in isolated myocardial trabeculae. RESULTS: LAMP-2 deficient mice displayed reduced ejection fraction (EF) (58.9+/-3.4 vs. 80.7+/-5.1%, P<0.05) and reduced cardiac output (8.3+/-3.1 vs. 14.7+/-3.6 ml/min, P<0.05) as compared to wild-type controls. Isolated multicellular muscle preparations from LAMP-2 deficient mice confirmed depressed force development (3.2+/-0.6 vs. 8.4+/-0.9 mN/mm2, P<0.01). All groups showed similar force-frequency behaviour when normalised to baseline force. Post-rest potentiation was significantly depressed at intervals>15 s in LAMP-2 deficient mice (P<0.05). Although attenuated in absolute force development, the normalised inotropic response to increased calcium and beta-adrenoreceptor stimulation was unaltered. Electron microscopic analysis revealed autophagic vacuoles in LAMP-2 deficient cardiomyocytes. Protein analysis showed unaltered levels of SERCA2a, calsequestrin and phospholamban. CONCLUSIONS: Cardiac contractile function in LAMP-2 deficient mice as a model for Danon disease is significantly attenuated. The occurrence of autophagic vacuoles in LAMP-2 deficient myocytes is likely to be causal for the depressed contractile function resulting in an attenuated cardiac pump reserve.

Animals↗

PubMed2006

Frequency-dependent contractile response of isolated cardiac trabeculae under hypo-, normo-, and hyperthermic conditions.

The body is from time to time exposed to nonnormothermic conditions; both hypo- and hyperthermia can occur as a result of external (environment) or internal (pathogens, allergens) stressors. To preserve life under hypo- and hyperthermic conditions, adequate perfusion of vital organs is mandated. Although cardiac output regulation under hyperthermic conditions has been studied, the mechanical response of basic contractile function of the myocardium itself is incompletely understood. Accordingly, we set out to test mechanical output of isolated myocardium under hyperthermic conditions and to compare the results with the hypo- and normothermic response in the same tissue. We observed that, in absence of a frequency change, developed force decreased markedly. At a physiological normal stimulation rate of 6 Hz, developed force decreases to 67.2 +/- 2.6% at 42 degrees C compared with 37 degrees C. In addition, twitch timing characteristics also accelerate, allowing for a faster relaxation; time from peak tension to 50% relaxation is approximately 23% faster (from 31.4 +/- 2.6 to 24.4 +/- 1.7 ms). Although this faster relaxation in turn prevents a steep increase in diastolic tension at high frequencies, the very fast calcium kinetics now prevent a more complete activation of the myofilaments, resulting in a lower twitch-force maximum at hyperthermic conditions. Even at maximal beta-adrenergic stimulation, developed force is well below levels reached at physiological temperature.

Adrenergic beta-Agonists↗

PubMed2006

Murine strain differences in contractile function are temperature- and frequency-dependent.

Despite the widespread use of mice in the investigation of cardiac function, little is known as to what extent cardiac contractile function varies between different murine strains. We have investigated basic contractile function in isolated multicellular right ventricular trabeculae from three commonly used mouse strains (C57BL/6, SV129, and FVBN). Suitable trabeculae (<100 microm thick, >1 mm long) occurred rather frequently in FVBN and SV129 mice (on average about 2 per heart), but only sporadically in C57BL/6 mice (on average only 1 per 3-4 mice). However, using similar sized preparations under experimental conditions closely mimicking those in vivo (37 degrees C and frequencies between 8 and 12 Hz), contractile function was virtually identical. In addition, the magnitude of response to beta-adrenergic stimulation was also indistinguishable between the strains. However, at subphysiological frequency, FVBN mice consistently displayed more developed force compared to C57/BL6 and SV129 mice. Furthermore, contractile performance at a subphysiological temperature and frequency, where studies on isolated myocardium often are performed, was also strain-dependent. We conclude that basic contractile function under near physiological conditions is preserved throughout various strains, but subphysiological conditions can have a profound effect on contractile performance. Hence, choice of strain can have important implications for cardiac contractile function under nonphysiological conditions.

Animals↗

PubMed2006

Measurement of myofilament calcium sensitivity at physiological temperature in intact cardiac trabeculae.

Cardiac contraction-relaxation coupling is determined by both the free intracellular calcium concentration ([Ca2+]i) and myofilament properties. We set out to develop a technique where we could assess these parameters (twitch and steady-state force [Ca2+]i) under near physiological conditions. Bis-fura-2 was iontophorically introduced into ultrathin rat trabeculae preparations to monitor the [Ca2+]i, and steady-state contractures were achieved by using a modified Krebs-Henseleit solution containing high K+. During K+ contractures, the very slow changes in [Ca2+]i and force development were in equilibrium and allowed for the construction of a steady-state, force-[Ca2+]i relationship. Twitch contractions before and after this myofilament calcium sensitivity assessment were unaltered, and this protocol could be repeated several times. For the first time, this novel protocol allows us to measure myofilament calcium sensitivity under physiological temperature. Not only do the data so obtained allow us to assess myofilament calcium sensitivity, the data also will allow us, in the same preparation under nearly identical conditions, to compare the dynamic to the steady-state, force-calcium relationship. To test whether the steady-state relationship between force and calcium in our novel protocol reproduces expected changes, we determined this relationship in the presence of isoproterenol and under acidosis and alkalosis. As expected, beta-adrenergic stimulation resulted in an increase of calcium amplitude and twitch force and a desensitization of the myofilaments as indicated by a rightward shift of the obtained steady-state, force-calcium relationship. An increase in pH shifted the curve leftward, whereas a decrease in pH resulted in the expected rightward shift.

Actin Cytoskeleton↗

PubMed2005

Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.

Animals↗

PubMed2005

Effect of muscle dimensions on trabecular contractile performance under physiological conditions.

Multicellular cardiac muscles are widely used to study cardiac (patho-)physiology in vitro. One of the potential pitfalls of such experiments is that muscles with a large diameter have a larger diffusion barrier for transport of oxygen and waste products and can thus potentially form a hypoxic core. Although a sufficiently small muscle size is critical for obtaining unambiguous data, the relationship between muscle diameter and contractile performance specifically under near-physiological conditions remains unknown. Small uniform trabeculae of various diameters isolated from LBNF1 rats were stimulated at different temperatures (27.5-37.5 degrees C) and frequencies (1-8 Hz). Twitch contractions and rapid cooling contractures were used to assess contractile performance and SR Ca2+ load, respectively. We observed that at physiological frequencies and temperatures, contractile performance was clearly diminished in muscles with diameter >150 microm, likely due to the decreased rates of oxygen supply and waste removal. At room temperature slower contractions allow sufficient time for oxygen diffusion into the muscle core, and as a result the difference in contractile performance between the thin and thick muscles was less. Thus, in order to exclude adverse effects on contractile performance in multicellular myocardium under physiological conditions, it is essential that the preparations are of sufficient small diameter (<0.15 mm).

Animals↗

PubMed2005

Utrophin deficiency worsens cardiac contractile dysfunction present in dystrophin-deficient mdx mice.

The loss of dystrophin in patients with Duchenne muscular dystrophy (DMD) causes devastating skeletal muscle degeneration and cardiomyopathy. Dystrophin-deficient (mdx) mice have a much milder phenotype, whereas double knockout (DKO) mice lacking both dystrophin and its homolog, utrophin, exhibit the clinical signs observed in DMD patients. We have previously shown that DKO and mdx mice have similar severities of histological features of cardiomyopathy, but no contractile functional measurements of DKO heart have ever been carried out. To investigate whether DKO mice display cardiac dysfunction at the tissue level, contractile response of the myocardium was tested in small, unbranched, ultrathin, right ventricular muscles. Under near physiological conditions, peak isometric active developed tension (F(dev), in mN/mm2) at a stimulation frequency of 4 Hz was depressed in DKO mice (15.3 +/- 3.7, n = 8) compared with mdx mice (24.2 +/- 5.4, n = 7), which in turn were depressed compared with wild-type (WT) control mice (33.2 +/- 4.5, n = 7). This reduced Fdev was also observed at frequencies within the murine physiological range; at 12 Hz, Fdev was (in mN/mm2) 11.4 +/- 1.8 in DKO, 14.5 +/- 4.2 in mdx, and 28.8 +/- 5.4 in WT mice. The depression of Fdev was observed over the entire frequency range of 4-14 Hz and was significant between DKO versus mdx mice, as well as between DKO or mdx mice versus WT mice. Under beta-adrenergic stimulation (1 micromol/l isoproterenol), Fdev in DKO preparations was only (in mN/mm2) 14.7 +/- 5.1 compared with 30.9 +/- 8.9 in mdx and 41.0 +/- 4.9 in WT mice. These data show that cardiac contractile dysfunction of mdx mice is generally worsened in mice also lacking utrophin.

Animals↗

PubMed2004

Improved systolic and diastolic myocardial function with intracoronary pyruvate in patients with congestive heart failure.

BACKGROUND: Pyruvate increases myocardial performance in isolated myocardium and improves hemodynamics in patients with congestive heart failure. AIMS: To investigate the influence of pyruvate on detailed parameters of systolic and diastolic left ventricular (LV) function. METHODS AND RESULTS: In patients with heart failure due to dilated cardiomyopathy (LVEF 30+/-4%, n=9) pyruvate was infused intracoronarily. LV function was analysed before, during and after application of different pyruvate concentrations using a LV-micromanometer catheter. LV volumes were determined using cine ventriculography. Pyruvate increased maximum rate of LV isovolumic pressure rise (Peak +dP/dt) from 802+/-106 to 1125+/-103 mmHg/s (P<0.05). Left ventricular end-diastolic pressure declined in parallel from 17+/-2 to 12+/-2 mmHg (P<0.05) and heart rate decreased from 79+/-4 to 72+/-5 min(-1) (P<0.05). Stroke volume index increased from 34+/-4 to 43+/-6 ml/m(2) (P<0.05), end-diastolic LV volume remained unchanged, thus left ventricular ejection fraction increased with pyruvate from 30+/-4 to 39+/-4% (P<0.05). Maximum rate of LV isovolumic pressure decline (Peak -dP/dt) was significantly increased with pyruvate (from 794+/-94 to 980+/-108 mmHg/s; P<0.05) and mean arterial pressure increased from 80+/-5 to 88+/-4 mmHg (P<0.05). Discontinuation of pyruvate resulted in immediate reversibility of its effects. CONCLUSION: Intracoronary pyruvate improves systolic and diastolic myocardial function and increases ejection fraction without increasing heart rate. Pyruvate thus exhibits the profile of a favourable inotropic agent, however, further investigation for the treatment of patients with acute heart failure is mandatory.

Adult↗

PubMed2004

Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites.

Acute beta-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD22,23). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (P<0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD22,23 (P<0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.

Actin Cytoskeleton↗

PubMed2004

Abnormal frequency-dependent responses represent the pathophysiologic signature of contractile failure in human myocardium.

BACKGROUND: - The normal increase in isometric developed force (DF) with faster pacing rates, known as the positive force-frequency response/relationship (FFR), is altered in failing myocardium, as shown by its negative response to increased pacing. The objective of this study was to determine if increasing Ca2+ influx with L-type Ca2+ channel (L-CaCh) agonists: BayK 8644 (BayK) and FPL 64176 (FPL) or increased extracellular Ca2+ could increase contractility and normalize the FFR in failing myocardium. METHODS: - Isometric DF was measured in right ventricular trabeculae from failing (n = 28) and non-failing (n = 12) human hearts at various stimulation frequencies (0.5-2.5 Hz) before and after bath application of BayK (250 nM), FPL (100 nM), or high Ca2+ (7.0 mM). Post-rest (PR) experiments were also conducted on several trabeculae. RESULTS: - In trabeculae from failing hearts, the DF decreased with an increase in pacing. Addition of L-CaCh agonists increased DF to similar levels in trabeculae from both failing and non-failing hearts at slow pacing rates, but did not alter the negative FFR in the failing group. During increased rest intervals, the amount of PR potentiation was diminished in trabeculae from failing hearts as compared to the non-failing preparations. CONCLUSION: - This study demonstrates that the abnormal FFR observed in trabeculae from failing hearts is a reliable physiologic signature of the cardiomyopathic state even when DF, at slow stimulation frequencies, is relatively high. These studies further demonstrate that the impaired FFR is not due to an inability to further increase contractility. Rather, our findings suggest that the abnormal FFR and blunted PR potentiation alike are a reflection of an altered functional balance between Ca2+ re-uptake and Ca2+ extrusion.

Calcium↗

PubMed2004

Cardiac adenoviral S100A1 gene delivery rescues failing myocardium.

Cardiac-restricted overexpression of the Ca2+-binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load mechanistically due to increased SR Ca2+ uptake and reduced SR Ca2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na+ concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.

Adenoviridae↗

PubMed2002

Selective contractile dysfunction of left, not right, ventricular myocardium in the SHHF rat.

The progression of hypertension to cardiac failure involves systemic changes that may ultimately affect contractility throughout the heart. Spontaneous hypertensive heart failure (SHHF) rats have depressed left ventricular (LV) function, but right ventricular (RV) dysfunction is less well characterized. Ultrathin (87 +/- 5 mircom) trabeculae were isolated from end-stage failing SHHF rats and from age-matched controls. Under near-physiological conditions (1 mM Ca(2+), 37 degrees C, 4 Hz), developed force (in mN/mm(2)) was not significantly different in SHHF LV and RV trabeculae and those of controls. SHHF LV preparations displayed a negative force-frequency behavior (40 +/- 7 vs. 23 +/- 4 mN/mm(2), 2 vs. 7 Hz); this relationship was positive in SHHF RV preparations (27 +/- 5 vs. 40 +/- 6 mN/mm(2)) and controls (32 +/- 6 vs. 44 +/- 9 mN/mm(2)). The response to isoproterenol (10(-6) M, 4 Hz) was depressed in SHHF LV preparations. The inotropic response to hypothermia was lost in SHHF LV trabeculae but preserved in SHHF RV trabeculae. Intracellular calcium measurements revealed impaired calcium handling at higher frequencies in LV preparations. We conclude that in end-stage failing SHHF rats, RV function is only marginally affected, whereas a severe contractile dysfunction of LV myocardium is present.

Adrenergic beta-Agonists↗

PubMed2002

Intracellular beta-blockade: overexpression of Galpha(i2) depresses the beta-adrenergic response in intact myocardium.

OBJECTIVE: Increased levels of inhibitory G proteins have been observed in heart failure, but their physiological relevance in mediating the reduced beta-adrenergic response is largely unknown. METHODS: To evaluate the functional consequences of Galpha(i2) overexpression, we studied myocardial contraction in intact isometric contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of Galpha(i2). RESULTS: Neither Galpha(i2) nor lacZ (control) overexpression altered baseline contractile force. After 72 h of continuous contractions, developed force (F(dev)) increased after addition of 1 microM isoproterenol by 28.5+/-9.7 mN/mm(2) in the control group, which was unchanged from the initial response at t=0 h (23.7+/-3.8 mN/mm(2)). In sharp contrast, in preparations transfected with AdGalpha(i2), the response to isoproterenol was significantly attenuated (5.9+/-2.0 vs. 27.6+/-4.2 mN/mm(2), t=72 vs. 0 h, respectively, P<0.01). In a primary culture of transfected isolated myocytes from a nearly identical baseline, isoproterenol increased cell shortening by 3.1+/-0.6% in the lacZ transfected myocytes, but only by 1.3+/-0.5% in Galpha(i2) transfected myocytes (t=72 h, P<0.01). In Galpha(i2) transfected myocytes, pertussis toxin restored beta-adrenergic responsiveness, indicating specificity of attenuation by the transgene. CONCLUSIONS: Overexpression of Galpha(i2) attenuates the positive inotropic effects of beta-adrenergic stimulation in myocardium. In addition, the method we developed allows investigation of a causal link between altered protein expression and subsequent alterations in contractile function in a physiological relevant in vitro manner.

Adenoviridae↗

PubMed2002

Hydroxyl radical-induced acute diastolic dysfunction is due to calcium overload via reverse-mode Na(+)-Ca(2+) exchange.

Hydroxyl radicals (OH) are involved in the development of reperfusion injury and myocardial failure. In the acute phase of the OH-mediated diastolic dysfunction, increased intracellular Ca(2+) levels and alterations of myofilaments may play a role, but the relative contribution of these systems to myocardial dysfunction is unknown. Intact contracting cardiac trabeculae from rabbits were exposed to OH, resulting in an increase in diastolic force (F(dia)) by 540%. Skinned fiber experiments revealed that OH-exposed preparations were sensitized for Ca(2+) (EC(50): 3.27+/-0.24 x 10(-6) versus 2.69+/-0.15 x 10(-6) mol/L; P<0.05), whereas maximal force development was unaltered. Western blots showed a proteolytic degradation of troponin T (TnT) with intact troponin I (TnI). Blocking of calpain I by MDL-28.170 inhibited both TnT-proteolysis and Ca(2+) sensitization, but failed to prevent the acute diastolic dysfunction in the intact preparation. The OH-induced diastolic dysfunction was similar in preparations with intact (540+/-93%) and pharmacologically blocked sarcoplasmic reticulum (539+/-77%), and was also similar in presence of the L-type Ca(2+)-channel antagonist verapamil. In sharp contrast, inhibition of the reverse-mode sodium-calcium exchange by KB-R7943 preserved diastolic function completely. Additional experiments were performed in rat myocardium; the rise in diastolic force was comparable to rabbit myocardium, but Ca(2+) sensitivity was unchanged and maximal force development was reduced. This was associated with a degradation of TnI, but not TnT. Electron microscopic analysis revealed that OH did not cause irreversible membrane damage. We conclude that OH-induced acute diastolic dysfunction is caused by Ca(2+) influx via reverse mode of the sodium-calcium exchanger. Degradation of troponins appears to be species-dependent but does not contribute to the acute diastolic dysfunction.

Actin Cytoskeleton↗

PubMed2002

Influence of pyruvate on contractile performance and Ca(2+) cycling in isolated failing human myocardium.

BACKGROUND: Application of pyruvate was shown to improve contractile function in isolated animal myocardium and hemodynamics in patients with congestive heart failure. We assessed the influence of pyruvate on systolic and diastolic myocardial function and its subcellular mode of action in isolated myocardium from end-stage failing human hearts. METHODS AND RESULTS: In muscle strip preparations, concentration-dependent effects of pyruvate on developed and diastolic force (n=6), aequorin light emission reflecting intracellular Ca(2+) transients (n=6), and rapid cooling contractures reflecting sarcoplasmic reticulum (SR) Ca(2+) content (n=11) were measured. Pyruvate resulted in a concentration-dependent increase in developed force and a decrease in diastolic force, with a maximum effect of 155% and 21%, respectively, at 20 mmol/L pyruvate (P<0.05). This was associated with a dose-dependent prolongation of time to peak tension and relaxation time. Pyruvate increased rapid cooling contractures by 51% and aequorin light signals by 85% (at 15 and 20 mmol/L; P<0.05). This indicates increased SR Ca(2+) content and increased intracellular Ca(2+) transients. The inotropic effect of pyruvate was still present after elimination of SR Ca(2+) storage function with 10 micromol/L cyclopiazonic acid and 1 micromol/L ryanodine (n=8). Pyruvate significantly increased intracellular pH from 7.31+/-0.03 to 7.40+/-0.04 by BCECF fluorescence (n=6). CONCLUSIONS: The present findings indicate that pyruvate improves contractile performance of failing human myocardium by increasing intracellular Ca(2+) transients as well as myofilament Ca(2+) sensitivity. The former seem to result from increased SR Ca(2+) accumulation and release, the latter from increased intracellular pH.

Aequorin↗

PubMed2002

Physiological determinants of contractile force generation and calcium handling in mouse myocardium.

Despite the fact that the mouse has become a common tool to study cardiac dysfunction, little is known regarding the regulation of murine cardiac contractility. We have investigated the three main mechanisms that regulate cardiac output (frequency-dependent activation, length-dependent activation, and beta-adrenergic stimulation) in ultra-thin right ventricular (RV) trabeculae from the mouse heart at body temperature (37 degrees C). [Ca(2+)](i) was recorded in a subset of trabeculae iontophoretically loaded with fura-2, and rapid cooling contractures were performed to estimate the sarcoplasmic reticulum (SR) calcium load. The force-frequency relationship was positive (2-12Hz); force increased, albeit slightly, while relaxation timing decreased. As expected, in response to beta-adrenergic stimulation, force development increased while contractile duration decreased, and increased muscle length led to increased force generation. Changes in SR calcium load and the calcium transient amplitude paralleled effects on active force generation. Despite several qualitative similarities with other mammalian species, the reserve for augmentation of force via either increased frequency or beta-adrenergic stimulation was considerably smaller in mouse than in other animals. Therefore, changes in preload, as opposed to increased HR or adrenergic tone, appears to be a much more important determinant of cardiac performance in the mouse than in larger mammals.

Adrenergic beta-Agonists↗