Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.     

Calcium entry via Na/Ca exchange during the action potential directly contributes to contraction of failing human ventricular myocytes

Prolongation of the Ca2+ transient and action potential (AP) durations are two characteristic changes in myocyte physiology in the failing human heart. The hypothesis of this study is that Ca2+ influx via reverse mode Na+/Ca2+ exchanger (NCX) or via L-type Ca2+ channels directly activates contraction in failing human myocytes while in normal myocytes this Ca2+ is transported into the sarcoplasmic reticulum (SR) to regulate SR Ca2+ stores. METHODS: Myocytes were isolated from failing human (n=6), nonfailing human (n=3) and normal feline hearts (n=9) and whole cell current and voltage clamp techniques were used to evoke and increase the duration of APs (0.5 Hz, 37 degrees C). Cyclopiazonic acid (CPA 10(-6) M), nifedipine (NIF;10(-6) M) and KB-R 7943 (KB-R; 3x10(-6) M) were used to reduce SR Ca2+ uptake, Ca2+ influx via the L-type Ca2+ current and reverse mode NCX, respectively. [Na+)i was changed by dialyzing myocytes with 0, 10 and 20 mM Na(+) pipette solutions. RESULTS: Prolongation of the AP duration caused an immediate prolongation of contraction and Ca2+ transient durations in failing myocytes. The first beat after the prolonged AP was potentiated by 21+/-5 and 27+/-5% in nonfailing human and normal feline myocytes, respectively (P<0.05), but there was no significant effect in failing human myocytes (+5+/-4% vs. steady state). CPA blunted the potentiation of the first beat after AP prolongation in normal feline and nonfailing human myocytes, mimicking the failing phenotype. NIF reduced steady state contraction in feline myocytes but the potentiation of the first beat after AP prolongation was unaltered (21+/-3% vs. base, P<0.05). KB-R reduced basal contractility and abolished the potentiation of the first beat after AP prolongation (2+/-1% vs. steady state). Increasing [Na+]i shortened AP, Ca2+ transient and contraction durations and increased steady state and post AP prolongation contractions. Dialysis with 0 Na+ eliminated these effects. CONCLUSIONS: Ca2+ enters both normal and failing cardiac myocytes during the late portion of the AP plateau via reverse mode NCX. In (normal) myocytes with good SR function, this Ca(2+) influx helps maintain and regulate SR Ca2+ load. In (failing) human myocytes with poor SR function this Ca2+ influx directly contributes to contraction. These studies suggest that the Ca2+ transient of the failing human ventricular myocytes has a higher than normal reliance on Ca2+ influx via the reverse mode of the NCX during the terminal phases of the AP.     

Isolated ventricular myocytes from failing and non-failing human heart; the relation of age and clinical status of patients to isoproterenol response.

Single cardiac myocytes were isolated from the ventricles of failing and non-failing human hearts. The contraction amplitude, time-to-peak shortening and time to 50% and 90% relaxation were measured in cells stimulated at 0.2 Hz at 32 degrees C. The effects of increasing extracellular calcium and isoproterenol were investigated using cumulative concentration/response curves. Maximum contraction amplitude in high calcium or velocities of contraction or relaxation were not impaired in cells from failing hearts. Beta-adrenoceptor function in a single cell was assessed by the maximum contraction amplitude in the presence of isoproterenol relative to that with high calcium in the same cell (isoproterenol/calcium ratio). A decrease in the isoproterenol/calcium ratio correlated positively with an increase in the isoproterenol EC50 (concentration for half-maximal effect) for a cell (P less than 0.02, n = 39). The isoproterenol/calcium ratio in left ventricular myocytes decreased with increasing severity of disease, correlating with failure as defined by New York Heart Association class (P less than 0.001, n = 26 patients), left ventricular ejection fraction (P less than 0.001, n = 24), left ventricular end diastolic pressure (P less than 0.05, n = 21) and amount of diuretics prescribed (P less than 0.001, n = 26). In right ventricular myocytes, only increasing NYHA class correlated with decreasing isoproterenol/calcium ratios. There was a correlation of the isoproterenol/calcium ratio between right and left ventricular cells from patients with ischemic heart disease (P less than 0.05), n = 11). Beta-adrenoceptor subsensitivity occurred in mitral valve disease, ischemic heart disease, congenital abnormalities and congestive cardiomyopathy, but not in the right ventricle of patients with myocarditis. The isoproterenol/calcium ratio correlated negatively with the age of the patient (P less than 0.001, n = 26, left ventricle). Multiple regression indicated that the maximum contraction amplitudes in either high isoproterenol or high calcium declined significantly with age only, but that both age and severity of disease contributed to the decrease in isoproterenol/calcium ratio. Time-to-peak tension in isoproterenol, as well as relaxation times in high calcium also decreased with the age of the patient. Analysis of variance showed that between-patient variation was significantly greater than between-cell for most of the parameters measured. Beta-adrenoceptor desensitisation may be detected in individual myocytes from failing hearts, and this relates more to the severity of disease and the age of the patient rather than the etiology of heart failure. A decline in absolute contractility of muscle cells with age was detected.     

Evidence for a sustained effectiveness of sodium-channel activators in failing human myocardium.

Elevation of cytosolic sodium is thought to be correlated with an increase in force of contraction due to an activation of sodium-calcium exchange. We investigated the inotropic response mediated by the new sodium-channel activator BDF 9148 (0.01-100 mumol/l) on failing human myocardium. Force of contraction was studied using electrically driven human papillary muscle strips from moderately (NYHA II-III, mitral valve replacement) and terminally (NYHA IV, heart transplantation) failing hearts. We also investigated the effects in auricular trabeculae from non-failing hearts (aortocoronary bypass operation). Results were compared with inotropic responses to DPI 201-106 (DPI, 0.1-3 mumol/l), Ca2+ (1.8-15 mmol/l) and isoprenaline (0.001-1 mumol/l). Carbachol (100 mumol/l) and adenosine (1000 mumol/l) were examined in the presence of BDF 9148 and isoprenaline. Both sodium-channel activators, BDF 9148 and DPI 201-106, increased force of contraction in a dose-dependent manner in papillary muscle strips as well as in auricular trabeculae. BDF 9148 and DPI 201-106 were more effective (max. PIE NYHA II-III 1.6 +/- 0.2 mN, NYHA IV 5.9 +/- 0.7 mN, P less than 0.05) and more potent (EC50 (in mumol/l): NYHA IV 0.35, 0.19-0.66; NYHA II-III 1.85, 1.37-2.41) in terminally failing as compared to moderately failing left ventricular myocardium. Moreover, the positive inotropic effects of BDF 9148 were greater than those of DPI 201-106 in NYHA IV (max. PIE 2.7 +/- 0.3 mN, P less than 0.05). In NYHA IV, BDF 9148 was as effective as CA2+ (max. PIE 5.1 +/- 0.4 mN). In the same hearts, the positive inotropic effects of isoprenaline were reduced in NYHA IV (max. PIE 2.1 +/- 0.3 mN) compared to NYHA II-III (max. PIE 3.4 +/- 0.4 mN, P less than 0.05). Adenosine as well as carbachol did not affect the positive inotropic response of BDF 9148 or DPI 201-106 but reduced the effectiveness of isoprenaline (P less than 0.05). In myocardial membranes, BDF 9148 was 1000-fold less effective in competition experiments with 3H-ouabain than ouabain. We conclude that (1) sodium-channel activators may produce a significant cAMP-independent positive inotropic effect in left ventricular myocardium from failing human hearts; (2) the inotropic effect of sodium-channel activators were more potent and more effective in NYHA IV as compared to NYHA II-III. The degree of myocardial failure does not reduce the effectiveness of the sodium-channel activator BDF 9148.     

Troponin phosphorylation and regulatory function in human heart muscle: dephosphorylation of Ser23/24 on troponin I could account for the contractile defect in end-stage heart failure

We made quantitative measurements of phosphorylation in troponin isolated from 6 non-failing donor hearts and 6 explanted hearts with end-stage heart failure in SDS-PAGE gels using Pro-Q Diamond phosphoprotein stain. The troponin T phosphorylation level was the same in troponin from failing and non-failing heart (3.1 mol Pi/mol). However, troponin I phosphorylation was significantly lower in failing (0.37+/-0.18 mol Pi/mol) compared with non-failing heart troponin (2.25+/-0.36 mol Pi/mol). Levels of troponin I PKA-dependent phosphorylation, measured with a phosphoserine 23/24-specific antibody, were also significantly lower in failing heart troponin (0.19+/-0.06 mol Pi/mol) compared to non-failing troponin (1.14+/-0.09 mol Pi/mol). We calculate that there is phosphorylation in addition to serine 23/24 of 1.11+/-0.34 mol Pi/mol in non-failing reduced to 0.18+/-0.17 mol Pi/mol in failing heart troponin, attributed to phosphorylation on the PKC sites. To test for the functional role of troponin I phosphorylation, the native troponin I from either non-failing or failing heart troponin was exchanged for a recombinant (unphosphorylated) human cardiac troponin I. Thin filament Ca(2+)-regulatory function was studied with the quantitative in vitro motility assay: thin filaments containing the replaced troponin I resulted in a failing phenotype of a 17-26% reduced sliding speed and an increased Ca(2+)-sensitivity relative to non-failing troponin (EC(50) TnI-exchanged/non-failing=0.57, p<0.001). When exchanged with troponin I phosphorylated with PKA motility parameters reverted to a pattern indistinguishable from non-failing troponin (p=0.35-0.75). We suggest that changes in troponin function can account for the contractile abnormality in failing heart muscle and that the functional changes in troponin are due to reduced phosphorylation of troponin I at the PKA sites.     

Positive inotropic effects of NO donors in isolated guinea-pig and human cardiomyocytes independent of NO species and cyclic nucleotides

OBJECTIVE: To characterise the inotropic response of isolated myocytes to a range of structurally unrelated NO donors and to assess the role of NO release kinetics, NO species and cyclic nucleotides in mediating the observed changes. METHOD: Guinea-pig (GP) and human myocytes were prepared by enzymatic digestion. Paced contractile amplitude was recorded at 37 degrees C. NO release was measured by reduction of oxyhaemoglobin and using an NO electrode. Cyclic nucleotides were measured using a tritium labelled competitive binding assay. RESULTS: The NO donors S-nitrosoglutathione (GSNO) and diethylamine/NO (DEA/NO) produced positive inotropic effects in GP myocytes at (10(-5) M) (25 and 111% increases of contraction amplitude).The response to GSNO was significantly enhanced in the presence of a low concentration of isoprenaline (3x10(-10) M). Positive inotropy was observed with a range of both thiol and non-thiol donors, amongst which a fast rate of NO release was associated with positive inotropy. The response to GSNO was abolished by the free NO scavenger oxyhaemoglobin, but not by ODQ (soluble guanylyl cyclase [sGC] inhibitor), Rp-cAMPS (protein kinase A inhibitor) or thapsigargin (sarcoplasmic reticulum Ca(2+) uptake blocker). Direct measurement of cyclic nucleotides showed a rise in cGMP but not cAMP. Human ventricular myocytes showed a significant increase of contraction with GSNO (48+/-15.8%, n=7, P<0. 05) in the presence of isoprenaline and a marked response to DEA/NO alone. CONCLUSIONS: Isolated GP and human myocytes show a positive inotropic effect with certain NO donors. This is independent of sGC and cAMP. The rate of NO release from donors appears important in mediating the effect.     

Alterations of cross-bridge kinetics in human atrial and ventricular myocardium

CONDENSED ABSTRACT: We analyzed actomyosin cross-bridge kinetics in human atrial and ventricular muscle strip preparations by using sinusoidal length changes from 0.1 to 60 Hz. The minimum stiffness frequency was higher in atrial than in ventricular human myocardium and lower in failing than in non-failing left ventricular human myocardium. beta-Adrenergic stimulation increased the minimum stiffness frequency by 18 +/- 3% (p < 0.05). Cross-bridge kinetics are temperature-dependent, with a Q10 of at least 2.7. BACKGROUND: Dynamic stiffness measurements have revealed acute and chronic alterations of actomyosin cross-bridge kinetics in cardiac muscles of a variety of different animal species. We studied dynamic stiffness in right atrial and left ventricular preparations of non-failing and failing human hearts and tested the influence of the temperature and beta-adrenergic stimulation on cross-bridge kinetics. METHODS AND RESULTS: Muscle strips were prepared from right atria and left ventricles from human non-failing and failing hearts. After withdrawal of calcium, steady contracture tension was induced by the addition of 1.5 mM barium chloride. Sinusoidal length oscillations of 1% muscle length were applied, with a frequency spectrum of between 0.1 and 60 Hz. Dynamic stiffness was calculated from the length change and the corresponding force response amplitude. The specific minimum stiffness frequency, which indicates the interaction between cross-bridge recruitment and cross-bridge cycling dynamics, was analyzed for each condition: (1) The minimum stiffness frequency was 0.78 +/- 0.04 Hz in left ventricular myocardium and 2.80 +/- 0.31 Hz in right atrial myocardium (p < 0.01) at 27 degrees C. (2) The minimum stiffness frequency was 41% higher in non-failing compared to failing left ventricular human myocardium. (3) Over a wide range of experimental temperatures, the minimum stiffness frequency changed, with a Q10 of at least 2.7. (4) beta-Adrenergic stimulation significantly (p < 0.05) increased the minimum stiffness to 18 +/- 3% higher frequencies and significantly (p < 0.05) lowered contracture tension by 7 +/- 1%. CONCLUSIONS: The contractility of human heart muscle is not only regulated by excitation-contraction coupling but also by modulation of intrinsic properties of the actomyosin system. Acute and chronic alterations of cross-bridge kinetics have been demonstrated, which play a significant role in the physiology and pathophysiology of the human heart.     

Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite

OBJECTIVES: This study examined the effects of endotoxin on cardiac contractility in human myocardium. BACKGROUND: In animal myocardium, endotoxin and cytokine treatment led to enhanced inducible nitric oxide synthase (iNOS) expression and contractile dysfunction. Effects in human myocardium are unknown. METHODS: Left ventricular myocardial preparations from failing (n = 18) and nonfailing (n = 5) human hearts were incubated for 6 and 12 h in tyrode solution or in tyrode plus lipopolysaccharides (LPS), with LPS plus N(G)-mono-methyl-L-arginine (L-NMMA), with LPS plus hemoglobin or with LPS plus the superoxide scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). Force of contraction in response to isoprenaline (0.001 to 3 micromol/liter) was determined in electrically stimulated muscle preparations. The iNOS mRNA expression was examined by in situ hybridization and by polymerase chain reaction. The cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay. RESULTS: Isoprenaline concentration dependently increased force of contraction. Six and 12 hours of LPS treatment of failing myocardium decreased maximum inotropic response to isoprenaline by 54% (p = 0.009) and by 69% (p = 0.0023), respectively. In nonfailing myocardium, 12 h of LPS treatment decreased maximum inotropic effect of isoprenaline by 66% (p < 0.001). The LPS effects were attenuated by L-NMMA, hemoglobin and also Tiron. The iNOS mRNA was expressed in all LPS-treated preparations but also in most control myocardial preparations. In situ hybridization revealed iNOS expression within cardiac myocytes. There was no increase in myocardial cGMP content in response to endotoxin. CONCLUSIONS: Endotoxin exposure of human myocardium leads to a depression of cardiac contractility, which is mediated by enhanced iNOS activity and release of nitric oxide (NO). Consecutive reaction of NO with superoxide and formation of peroxynitrite may contribute to the decrease in force of contraction.     

In vitro motility analysis of thin filaments from failing and non-failing human heart: troponin from failing human hearts induces slower filament sliding and higher Ca(2+) sensitivity

Contractility of the myocardium is altered in end-stage heart failure. We investigated whether this was related to functional changes in troponin. We isolated troponin from 1 g samples of end-stage failing, non-failing and foetal human heart and studied its regulation of actin-tropomyosin movement over immobilised HMM by in vitro motility assay. At pCa5.4 the sliding velocity of thin filaments reconstituted with non-failing heart troponin was 52+/-4% more than actin-tropomyosin, with failing heart troponin velocity increased by 35+/-2% and with foetal heart troponin velocity increased by 11+/-4%. Thin filaments containing troponin from failing hearts were more Ca(2+)-sensitive than non-failing heart troponin. EC(50) for the fraction of filaments motile and filament velocity decreased 1.76+/-0.20 and 1.89+/-0.62-fold respectively relative to non-failing heart troponin. With foetal heart troponin the EC(50) decreased 2.16+/-0.23 and 3.50+/-1.73-fold for fraction and velocity respectively. Western blots revealed no difference in troponin T or troponin I isoform expression in troponin from failing and non-failing adult hearts but foetal isoforms of troponin I and T were observed in troponin from foetal heart. The level of PKA phosphorylation of troponin from failing and non-failing heart was not significantly different, however, complete non-specific dephosphorylation of troponin abolished most of the difference between failing and non-failing heart troponin. These findings show functional alterations in troponin in failing hearts which could account for the reduced contractile function but there is no change in troponin isoform expression or PKA phosphorylation. Differential phosphorylation by other kinases may account for altered troponin function.     

Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function

In heart failure (HF) a main factor in reduced contractility is reduced SR Ca2+ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca2+-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with beta-galactosidase adenovirus as control) could enhance SERCA function and restore Ca2+ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca2+ transient amplitude (2.0+/-0.1 versus 1.6+/-0.05 F/Fo at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adbetagal had negative FFR. Ca2+ transients declined faster in AdPLB-dn versus Adbetagal myocytes (RT50%: 317+/-29 versus 551+/-90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca2+ load (3.5+/-0.3 versus 2.6+/-0.2 F/Fo at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca2+ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction.     

Regional expression and functional characterization of the L-type Ca2+-channel in myocardium from patients with end-stage heart failure and in non-failing human hearts

The purpose of the present study was to investigate the expression and functional relevance of sarcolemmal L-type Ca2+-channels in failing and non-failing human myocardium. The protein expression of sarcolemmal L-type Ca2+-channels was determined with 3H-(+)-PN 200-110-binding experiments and Western blot analysis using a specific antibody against the alpha1-subunit in membrane preparations of ventricular and atrial myocardium from both failing (n = 15) and non-failing hearts (n = 8). The gene expression of the ion conducting pore of the L-type Ca2+-channel was examined with Northern blot technique in human failing and non-failing RNA. For normalization the RNA expression of calsequestrin was used. In electrically driven ventricular papillary muscle strips and auricular trabeculae, the responses to nifedipine and Ca2+ as parameters of myocardial function were studied. The protein expression as measured by 3H-(+)-PN 200-110-binding (Bmax) and Western Blot analysis with calsequestrin as reference was similar in left ventricular failing and non-failing myocardium. However, both were reduced in atrial compared to ventricular tissue in failing and non-failing hearts. The KD remained unchanged. Calsequestrin levels were unaltered in failing and non-failing hearts. The gene expression of the alpha1-subunit was similar in human failing and non-failing hearts. The L-type Ca2+-channel antagonist nifedipine reduced force of contraction with the same potency and efficiency in ventricular failing and non-failing myocardium. In contrast, the potency of nifedipine was higher in atrial than in ventricular tissue. Consistently, atrial myocardium from patients with dilated cardiomyopathy was more sensitive towards Ca2+ than those of the control group. In conclusion, the altered Ca2+-homeostasis in failing human myocardium may be less due to changes in sarcolemmal L-type Ca2+-channel expression or function than due to an altered intracellular Ca2+-handling.     

Force/shortening-frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts.

BACKGROUND: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. METHODS AND RESULTS: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; n = 11) and nonfailing (donor hearts; n = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5-2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). CONCLUSIONS: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.     

Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKIIdelta. Here we acutely overexpress CaMKIIdeltaC using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKIIdeltaC transgenic mice. CaMKIIdeltaC protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKIIdeltaC versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (tau(0.5Hz)/tau(3Hz)=2.14+/-0.10 versus 1.87+/-0.10), and peak Ca2+ current (ICa) was increased by 31% (-7.1+/-0.5 versus -5.4+/-0.5 pA/pF, P<0.05). Ca2+ transient amplitude was not significantly reduced (-27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca2+ content (41%; P<0.05). Thus fractional SR Ca2+ release was increased by 60% (P<0.05). Diastolic SR Ca2+ leak assessed by Ca2+ spark frequency (normalized to SR Ca2+ load) was increased by 88% in CaMKIIdeltaC versus LacZ myocytes (P<0.05; in an multiplicity-of-infection-dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide. This enhanced SR Ca2+ leak may explain reduced SR Ca2+ content, despite measured levels of SR Ca2+-ATPase and Na+/Ca2+ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKIIdeltaC myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKIIdeltaC overexpression alters RyR function, leading to enhanced SR Ca2+ leak and reduced SR Ca2+ content but without reducing twitch contraction and Ca2+ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca2+ release in CaMKIIdeltaC myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.     

The sarcoplasmic reticulum and the Na+/Ca2+ exchanger both contribute to the Ca2+ transient of failing human ventricular myocytes

Our objective was to determine the respective roles of the sarcoplasmic reticulum (SR) and the Na+/Ca2+ exchanger in the small, slowly decaying Ca2+ transients of failing human ventricular myocytes. Left ventricular myocytes were isolated from explanted hearts of patients with severe heart failure (n=18). Cytosolic Ca2+, contraction, and action potentials were measured by using indo-1, edge detection, and patch pipettes, respectively. Selective inhibitors of SR Ca2+ transport (thapsigargin) and reverse-mode Na+/Ca2+ exchange activity (No. 7943, Kanebo Ltd) were used to define the respective contribution of these processes to the Ca2+ transient. Ca2+ transients and contractions induced by action potentials (AP transients) at 0.5 Hz exhibited phasic and tonic components. The duration of the tonic component was determined by the action potential duration. Ca2+ transients induced by caffeine (Caf transients) exhibited only a phasic component with a rapid rate of decay that was dependent on extracellular Na+. The SR Ca2+-ATPase inhibitor thapsigargin abolished the phasic component of the AP Ca2+ transient and of the Caf transient but had no significant effect on the tonic component of the AP transient. The Na+/Ca2+ exchange inhibitor No. 7943 eliminated the tonic component of the AP transient and reduced the magnitude of the phasic component. In failing human myocytes, Ca2+ transients and contractions exhibit an SR-related, phasic component and a slow, reverse-mode Na+/Ca2+ exchange-related tonic component. These findings suggest that Ca2+ influx via reverse-mode Na+/Ca2+ exchange during the action potential may contribute to the slow decay of the Ca2+ transient in failing human myocytes.     

Metabolic vasodilation in the human forearm is preserved in hypercholesterolemia despite impairment of endothelium-dependent and independent vasodilation

OBJECTIVE: Hypercholesterolemia has been shown to impair endothelium-mediated, nitric oxide (NO)-dependent responses to acetylcholine (ACh), serotonin, substance P and flow-mediated dilation. We have recently shown that NO contributes to metabolic vasodilation in the human forearm. We sought to determine whether metabolic vasodilation is impaired in healthy subjects with hypercholesterolemia. METHODS: We compared the forearm blood flow (FBF) responses to isotonic exercise, ACh and the endothelium-independent vasodilator sodium nitroprusside in young, otherwise healthy volunteers with hypercholesterolemia and controls before and after the NO inhibitor NG-monomethyl-L-arginine (L-NMMA). FBF was measured using venous occlusion plethysmography. Hypercholesterolemic (n = 20) and control (n = 20) subjects were age- and gender-matched. RESULTS: Total cholesterol (6.9 +/- 0.3 vs. 4.6 +/- 0.1 mmol/l, P < 0.0001), low density lipoprotein (4.9 +/- 0.4 vs. 2.7 +/- 0.1 mmol/l, P < 0.001) and triglyceride (1.3 +/- 0.2 vs. 0.8 +/- 0.1 mmol/l, P = 0.005) levels were higher in the hypercholesterolemic group. Basal FBF and resistance were similar in the two groups. Hypercholesterolemia impaired the peak FBF response to ACh (11.1 +/- 1.9 vs. 17.6 +/- 2.2 ml/100 ml/min, P = 0.03), and reduced the peak response to sodium nitroprusside (6.0 +/- 0.4 vs. 8.1 +/- 0.6 ml/100 ml/min, P < 0.01). However, hypercholesterolemia did not affect peak hyperemic FBF (13.1 +/- 1.0 vs. 13.2 +/- 1.0 ml/100 ml/min, P = 1.0) or the FBF volume repayment during the 1 or 5 min after exercise. Resting FBF was reduced by L-NMMA to a similar degree (by 33% vs. 40%, P = 0.17) in both groups. Although L-NMMA reduced peak hyperemic FBF (by 16% vs. 17%, P = 0.93) and the volume repaid after exercise in both groups, there were no differences between the two groups. CONCLUSIONS: Exercise-induced metabolic vasodilation is in part dependent on NO release. Hypercholesterolemia impairs NO-mediated vasodilation, but is not associated with a reduction in exercise-induced hyperemia. This may indicate that multiple compensatory mechanisms are operative in skeletal muscle metabolic vasodilation.     

Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.     

Increased phospholamban phosphorylation limits the force-frequency response in the MLP-/- mouse with heart failure

Reduced Ca(2+) release from the sarcoplasmic reticulum (SR) and a negative force-frequency relation characterize end-stage human heart failure. The MLP(-/-) mouse with dilated cardiomyopathy is used as a model to explore novel therapeutic interventions but the alterations in Ca(2+) handling in MLP(-/-) remain incompletely understood. We studied [Ca(2+)](i) in left ventricular myocytes from MLP(-/-) and WT mice (3-4 months old; whole-cell voltage clamp, 30 degrees C). At 1 Hz stimulation, the amplitude of [Ca(2+)](i) transients was similar. However, in contrast to WT, at higher frequencies the [Ca(2+)](i) transient amplitude declined in MLP(-/-) and there was no increase in SR Ca(2+) content. Unexpectedly, the decline of [Ca(2+)](i) was faster in MLP(-/-) than in WT (at 1 Hz, tau of 80 +/- 9 vs. 174 +/- 29 ms, P < 0.001) and the frequency-dependent acceleration of the decline was abolished suggesting an enhanced basal SERCA activity. Indeed, the Ca(2+) affinity of SR Ca(2+) uptake in homogenates was higher in MLP(-/-), with the maximal uptake rate similar to WT. Phosphorylation of phospholamban in MLP(-/-) was increased (2.3-fold at Ser(16) and 2.9-fold at the Thr(17) site, P < 0.001) with similar SERCA and total phospholamban protein levels. On increasing stimulation frequency to 4 Hz, WT, but not MLP(-/-), myocytes had a net gain of Ca(2+), suggesting inadequate Ca(2+) sequestration in MLP(-/-). In conclusion, increased baseline phosphorylation of phospholamban in MLP(-/-) leads to a reduced reserve for frequency-dependent increase of Ca(2+) release. This represents a novel paradigm for altered Ca(2+) handling in heart failure, underscoring the importance of phosphorylation pathways.     

Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies

Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca2+ handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca2+ transients with reduced peaks, but durations approximately 3-fold longer than controls. To discriminate between action potential effects on Ca2+ kinetics and direct changes in Ca2+ regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca2+ removal (tauCa). tauCa was prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (tauCa control, 216+/-25 ms, n=17; tauCa failing, 292+/-23 ms, n=22; P<0.05), but this difference was markedly accentuated when Na+/Ca2+ exchange was eliminated (tauCa control, 282+/-30 ms, n=13; tauCa failing, 576+/-83 ms, n=11; P<0. 005). Impaired sarcoplasmic reticular (SR) Ca2+ uptake and a greater dependence on Na+/Ca2+ exchange for cytosolic Ca2+ removal was confirmed by inhibiting SR Ca2+ ATPase with cyclopiazonic acid, which slowed Ca2+ removal more in control than in failing myocytes. beta-Adrenergic stimulation of SR Ca2+ uptake in cells from failing hearts sufficed only to accelerate tauCa to the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na+/Ca2+ exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na+/Ca2+ exchange protein was increased 104% relative to controls. Thus, SR Ca2+ uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na+/Ca2+ exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease.     

Contribution of caveolin protein abundance to augmented nitric oxide signaling in conscious dogs with pacing-induced heart failure

Myocardial NO signaling appears elevated in heart failure (HF). Whether this results from increased NO production, induction of the high-output NO synthase (NOS)2 isoform, or changes in NOS regulatory pathways (such as caveolae) remains controversial. We tested the hypothesis that increased abundance of caveolin-3 and/or sarcolemmal caveolae contribute to increased NO signaling in pacing-induced HF. Abundance of caveolin-3 (0.59+/-0.08 versus 0.29+/-0.08 arbitrary units, P = 0.01) but not caveolin-1 was increased in HF compared with control conditions, assessed by Western blot. Additionally, transmission electron microscopy revealed increased caveolae (2. 7+/-0.4 versus 1.3+/-0.3 per micrometer myocyte membrane, P<0.005). The association between caveolin-3 and NOS3 at the sarcolemma and T tubules was unchanged in HF compared with control myocytes. The impact of NOS inhibition with L-N(G)-methylarginine hydrochloride (L-NMMA) on beta-adrenergic inotropy was assessed in conscious dogs before and after HF. In control dogs, dobutamine (5 microg. kg(-1) x min(-1)) increased +dP/dt by 36+/-7%, and this was augmented to 66+/-24% by 20 mg/kg L-NMMA (P = 0.04 versus without L-NMMA, n = 8) but not affected by 10 mg/kg L-NMMA (34+/-10%, P = NS; n = 8). In HF, dobutamine +dP/dt response was depressed (P<0.001 versus control), and increased concentrations were required to match control inotropic responses (10 to 15 microg. kg(-1) x min(-1), 48+/-7%). L-NMMA enhanced +dP/dt responses similarly at 10 mg/kg (61+/-17%, P = 0.02; n = 4) and 20 mg/kg (54+/-7%, P = 0.04; n = 7). Caveolin-3 abundance positively correlated with L-NMMA augmentation of dobutamine inotropic responses in HF (r = 0.9, P = 0.03; n = 4). Thus, in canine pacing-induced HF, expression of caveolin-3 and of sarcolemmal caveolae is increased. This increase is associated with augmented agonist-stimulated NO signaling, likely via a compartmentation effect.     

L-type Ca2+ channel alpha 1c subunit isoform switching in failing human ventricular myocardium

The objectives of this study were to determine the relative abundance of the L-type Ca channel alpha 1c IVS3 isoforms that result from alternative splicing in normal human ventricular myocytes and to measure the changes in isoform expression in end stage heart failure. METHODS: mRNA was isolated from left ventricular tissue and myocytes from non-failing and failing human hearts. RT-PCR with isoform-specific primers was used to obtain cDNAs that were then mutated for use in competitive PCR reactions. An RNase protection assay was also used to confirm the presence of one of the novel isoforms. RESULTS: Four different alpha 1c IVS3 isoforms were found in non-failing human ventricular myocytes using RT-PCR. Two isoforms contained exon 31 (termed IVS3A isoforms) and two isoforms contained exon 32 (termed IVS3B isoforms). One of these isoforms has not been observed previously and contains exon 31 and all but the last six base pairs of exon 32. In non-failing human ventricular myocytes the IVS3A isoform is 2.5 times more abundant than the IVS3B isoform. There were significant changes in the relative abundance of these isoforms in failing hearts, with the IVS3B isoform being twice as abundant as the IVS3A isoform. All isoforms were confirmed by RNase protection analysis. CONCLUSIONS: These experiments show that there are at least four L-type Ca channel mRNA isoforms in the normal human heart and that the relative abundance of these isoforms changes significantly in heart failure. These alpha 1c isoform changes in heart failure are associated with dysfunctional electromechanical disturbances, but the specific physiological role of each L-type Ca channel isoform in normal and failing hearts needs to be defined.