Masahito Miura

Increased von Willebrand Factor in the Endocardium as a Local Predisposing Factor for Thrombogenesis in Overloaded Human Atrial Appendage

OBJECTIVES We investigated immunoreactive von Willebrand factor (vWF), a platelet adhesion molecule, in the endocardial endothelium and its relationship to thrombogenesis in the human atrial appendage. BACKGROUND Intra-atrial thrombogenesis is generally thought to be induced by blood stasis in the atrial appendage involved with atrial fibrillation (AF). Little attention has been paid to alterations of the endocardial endothelium on which the thrombus develops. METHODS Atrial appendage tissue was obtained at heart surgery or at autopsy from AF and non-AF cardiac patients and from noncardiac patients. Immunohistochemistry for endothelial cell markers including vWF, CD31, CD34 and endothelial nitric oxide synthase (eNOS) and platelet glycoprotein Ib/IX or IIb/IIIa was performed and semiquantitatively graded. RESULTS In contrast to the apparent immunostaining for CD31, CD34 and eNOS, only focal or little immunoreactive vWF was seen in the endocardium of noncardiac patients. Immunoreactive vWF in the endocardial endothelium was increased in most cardiac patients, particularly in the left, but not in the right, atrial appendage of patients with mitral valvular disease, irrespective of whether AF was present. Platelet adhesion/thrombus formation in the endocardium was found in limited sites in which the overlying endothelium was deficient in eNOS and CD34. When warfarin-treated cases were excluded, there was a significant correlation between the immunohistochemical grade for vWF and the degree of platelet adhesion/thrombus formation in the endocardium. CONCLUSIONS Immunoreactive vWF in the endocardial endothelium was increased in overloaded human atrial appendage, which may be a local predisposing factor for intraatrial thrombogenesis.

Spatial Nonuniformity of Excitation–Contraction Coupling Causes Arrhythmogenic Ca2+ Waves in Rat Cardiac Muscle

Ca2+ waves underlying triggered propagated contractions (TPCs) are initiated in damaged regions in cardiac muscle and cause arrhythmias. We studied Ca2+ waves underlying TPCs in rat cardiac trabeculae under experimental conditions that simulate the functional nonuniformity caused by local mechanical or ischemic local damage of myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small jet of solution with a composition that reduces excitation–contraction coupling (ECC) in myocytes within that segment. The jet solution contained either caffeine (5 mmol/L), 2,3-butanedione monoxime (BDM; 20 mmol/L), or low Ca2+ concentration ([Ca2+]; 0.2 mmol/L). Force was measured with a silicon strain gauge and sarcomere length with laser diffraction techniques in 15 trabeculae. Simultaneously, [Ca2+]i was measured locally using epifluorescence of Fura-2. The jet of solution was applied perpendicularly to a small muscle region (200 to 300 &mgr;m) at constant flow. When the jet contained caffeine, BDM, or low [Ca2+], during the stimulated twitch, muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening normal regions outside the jet. Typical protocols for TPC induction (7.5 s-2.5 Hz stimulus trains at 23°C; [Ca2+]o=2.0 mmol/L) reproducibly generated Ca2+ waves that arose from the border between shortening and stretched regions. Such Ca2+ waves started during force-relaxation of the last stimulated twitch of the train and propagated (0.2 to 2.8 mm/sec) into segments both inside and outside of the jet. Arrhythmias, in the form of nondriven rhythmic activity, were induced when the amplitude of the Ca2+-wave was increased by raising [Ca2+]o. Arrhythmias disappeared rapidly when uniformity of ECC throughout the muscle was restored by turning the jet off. These results show, for the first time, that nonuniform ECC can cause Ca2+ waves underlying TPCs and suggest that Ca2+ dissociated from myofilaments plays an important role in the initiation of Ca2+ waves.

Ca2+ waves during triggered propagated contractions in intact trabeculae

Triggered propagated contractions (TPCs) starting from damaged regions travel along multicellular cardiac muscle preparations. We have reported that octanol (100 microM) inhibits TPCs. The inhibitory effect of octanol on propagation of TPCs could be due to an effect of octanol on Ca(2+)-induced Ca2+ release (CICR) mediated by Ca2+ diffusion inside the single cell or on the diffusion of Ca2+ from cell to cell via gap junctions (GJs). Therefore, we studied the regional changes in intracellular Ca2+ concentration ([Ca2+]i) during TPCs and the effect of octanol on the permeability of gap junctions (PGJ) in rat cardiac trabeculae. [Ca2+]i was measured using electrophoretically injected fura 2 and an image-intensified charge-coupled device camera. PGJ was calculated from the diffusion coefficient for fura 2 in trabeculae (Dtrab) and in the myoplasm (Dmyop). After 1- and 3-h superfusion with 100 microM 1-octanol, Dmyop showed no significant changes, whereas Dtrab was reduced significantly. Therefore, calculated PGJ was reduced from 4.15 x 10(-5) to 2.10 x 10(-5) and 0.86 x 10(-5) cm/s, respectively. The propagation velocity of the regional increases in [Ca2+]i during TPCs was constant, averaging 1.69 +/- 1.48 mm/s (range 0.34-5.47 mm/s, n = 10). These observations support the hypothesis that TPCs are initiated near the damaged ends of trabeculae and are propagated by CICR from the sarcoplasmic reticulum mediated by diffusion of Ca2+ through cells and from cell to cell through GJs.Triggered propagated contractions (TPCs) starting from damaged regions travel along multicellular cardiac muscle preparations. We have reported that octanol (100 μM) inhibits TPCs. The inhibitory effect of octanol on propagation of TPCs could be due to an effect of octanol on Ca2+-induced Ca2+ release (CICR) mediated by Ca2+ diffusion inside the single cell or on the diffusion of Ca2+from cell to cell via gap junctions (GJs). Therefore, we studied the regional changes in intracellular Ca2+ concentration ([Ca2+]i) during TPCs and the effect of octanol on the permeability of gap junctions ( P GJ) in rat cardiac trabeculae. [Ca2+]iwas measured using electrophoretically injected fura 2 and an image-intensified charge-coupled device camera. P GJ was calculated from the diffusion coefficient for fura 2 in trabeculae ( D trab) and in the myoplasm ( D myop). After 1- and 3-h superfusion with 100 μM 1-octanol, D myop showed no significant changes, whereas D trab was reduced significantly. Therefore, calculated P GJ was reduced from 4.15 × 10-5 to 2.10 × 10-5 and 0.86 × 10-5 cm/s, respectively. The propagation velocity of the regional increases in [Ca2+]iduring TPCs was constant, averaging 1.69 ± 1.48 mm/s (range 0.34-5.47 mm/s, n = 10). These observations support the hypothesis that TPCs are initiated near the damaged ends of trabeculae and are propagated by CICR from the sarcoplasmic reticulum mediated by diffusion of Ca2+ through cells and from cell to cell through GJs.

Microvolt T Wave Alternans in Human Cardiac Hypertrophy: Electrical Instability and Abnormal Myocardial Arrangement

T Wave Alternans and Hypertrophy. Introduction: Although T wave alternans (TWA) is a promising risk marker for myocardial electrical instability, it remains unclear how the presence of TWA is related to myocardial damage.

Sarcomere mechanics in uniform and non-uniform cardiac muscle: A link between pump function and arrhythmias

Starlings Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starlings Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.

Normal and oxidized low density lipoproteins accumulate deep in physiologically thickened intima of human coronary arteries.

Diffuse intimal thickening (DIT) that develops as a physiologic adaptation in the arterial wall has been implicated to have a predilection for atherosclerosis. We histologically investigated the lipid accumulation process in the human coronary DIT by focusing on the localization of normal and oxidized low-density lipoproteins (LDLs). Immunohistochemistry for apolipoprotein B 100 (a major apolipoprotein of LDL) and 8-iso-prostaglandin F2α (an oxidative product in LDL) showed substantial accumulation of oxidized relative to normal LDLs in the deep layers of DIT (52/139 segments). Subendothelial deposition of normal rather than oxidized LDLs, known as an early event of fatty streak formation, was less frequently found (13/139 segments). In contrast with fibrofatty lesions, lipid accumulation localized deep in DIT was characterized by fine lipid droplets scattered in the preserved tissue and by its association with neither macrophage accumulation nor apoptosis in the constituent cells. On the other hand, the deep intimal location of lipid accumulation clearly coincided with increased type I and type III collagen and elastic fibers but rarely with sulfated proteoglycans including decorin, which were all strongly expressed in advanced lesions. This lipid accumulation was found only in sites with DIT of more than 200 μm, occasionally extending to the inner media and involving neovessel formation around it. The presence of deep intimal lipid accumulation was associated with reduced endothelium-dependent relaxation to substance P in isolated coronary rings. These results suggest that normal and oxidized LDLs accumulate preferably in the nutritional border zone of established DIT involving local extracellular matrix alterations but independently of inflammatory or apoptotic processes. This may contribute to the functional and morphologic abnormalities seen in human coronary atherogenesis that progresses slowly with age.

Accumulation of risk markers predicts the incidence of sudden death in patients with chronic heart failure

Sudden death is common in chronic heart failure (CHF). Risk stratification is the first step for primary prevention.

Role of sarcomere mechanics and Ca2+ overload in Ca2+ waves and arrhythmias in rat cardiac muscle

Abstract:  Ca2+ release from the sarcoplasmic reticulum (SR) depends on the sarcoplasmic reticulum (SR) Ca2+ load and the cytosolic Ca2+ level. Arrhythmogenic Ca2+ waves underlying triggered propagated contractions arise from Ca2+ overloaded regions near damaged areas in the cardiac muscle. Ca2+ waves can also be induced in undamaged muscle, in regions with nonuniform excitation–contraction (EC) coupling by the cycle of stretch and release in the border zone between the damaged and intact regions. We hypothesize that rapid shortening of sarcomeres in the border zone during relaxation causes Ca2+ release from troponin C (TnC) on thin filaments and initiates Ca2+ waves. Elimination of this shortening will inhibit the initiation of Ca2+ waves, while SR Ca2+ overload will enhance the waves. Force, sarcomere length (SL), and [Ca2+]i were measured and muscle length was controlled. A small jet of Hepes solution with an extracellular [Ca2+] 10 mM (HC), or HC containing BDM, was used to weaken a 300 μm long muscle segment. Trains of electrical stimuli were used to induce Ca2+ waves. The effects of small exponential stretches on triggered propagatory contraction (TPC) amplitude and propagation velocity of Ca2+ waves (Vprop) were studied. Sarcomere shortening was uniform prior to activation. HC induced spontaneous diastolic sarcomere contractions in the jet region and attenuated twitch sarcomere shortening; HC+ butanedione monoxime (BDM) caused stretch only in the jet region. Stimulus trains induced Ca2+ waves, which started inside the HC jet region during twitch relaxation. Ca2+ waves started in the border zone of the BDM jet. The initial local [Ca2+]i rise of the waves by HC was twice that by BDM. The waves propagated at a Vprop of 2.0 ± 0.2 mm/sec. Arrhythmias occurred frequently in trabeculae following exposure to the HC jet. Stretch early during relaxation, which reduced sarcomere shortening in the weakened regions, substantially decreased force of the TPC (FTPC) and delayed Ca2+ waves, and reduced Vprop commensurate with the reduction FTPC. These results are consistent with the hypothesis that Ca2+ release from the myofilaments initiates arrhythmogenic propagating Ca2+ release. Prevention of sarcomere shortening, by itself, did not inhibit Ca2+ wave generation. SR Ca2+ overload potentiated initiation and propagation of Ca2+ waves.

Effect of Nonuniform Muscle Contraction on Sustainability and Frequency of Triggered Arrhythmias in Rat Cardiac Muscle

Background— Arrhythmias are benign or lethal, depending on their sustainability and frequency. To determine why lethal arrhythmias are prone to occur in diseased hearts, usually characterized by nonuniform muscle contraction, we investigated the effect of nonuniformity on sustainability and frequency of triggered arrhythmias. Methods and Results— Force, membrane potential, and intracellular Ca2+ concentration ([Ca2+]i) were measured in 51 rat ventricular trabeculae. Nonuniform contraction was produced by exposing a restricted region of muscle to a jet of 20 mmol/L 2,3-butanedione monoxime (BDM) or 20 &mgr;mol/L blebbistatin. Sustained arrhythmias (>10 seconds) could be induced by stimulus trains for 7.5 seconds only with the BDM or blebbistatin jet (100 nmol/L isoproterenol, 1.0 mmol/L [Ca2+]o, 24°C). During sustained arrhythmias, Ca2+ surges preceded synchronous increases in [Ca2+]i, whereas the stoppage of the BDM jet made the Ca2+ surges unclear and arrested sustained arrhythmias (n=6). With 200 nmol/L isoproterenol, 2.5 mmol/L [Ca2+]o, and the BDM jet, lengthening or shortening of the muscle during sustained arrhythmias accelerated or decelerated their cycle in both the absence (n=10) and presence (n=10) of 100 &mgr;mol/L streptomycin, a stretch-activated channel blocker, respectively. The maximum rate of force relaxation correlated inversely with the change in cycle lengths (n=14; P<0.01). Sustained arrhythmias with the BDM jet were significantly accelerated by 30 &mgr;mol/L SCH00013, a Ca2+ sensitizer of myofilaments (n=10). Conclusion— These results suggest that nonuniformity of muscle contraction is an important determinant of the sustainability and frequency of triggered arrhythmias caused by the surge of Ca2+ dissociated from myofilaments in cardiac muscle.

Interaction between sarcomere and mitochondrial length in normoxic and hypoxic rat ventricular papillary muscles

We hypothesized that the mitochondrial length may be altered according to changes in the sarcomere length, and that this relationship may be affected by exposure to hypoxia. Rat ventricular papillary muscles were isolated and immersed in normoxic or hypoxic solutions for 10 min. Sarcomeres of various lengths were obtained by fixing the papillary muscles in a slack or stretched state, or after exposure to a contracture solution containing saponin and CaCl(2). The mitochondrial length measured using electron microscopy significantly correlated to the length of the adjacent sarcomere in both the normoxic (n=767) and hypoxic (n=1145) groups (P<.0001). The slope of the regression line, however, was significantly less steep, and its intercept was significantly larger in the hypoxic group than in the normoxic group (analysis of covariance). When we analyzed the mitochondrial lengths among the three sarcomere-length subgroups (<1.5, 1.5-2.0, and >2.0 microm), the mitochondrial length was significantly shorter in the hypoxic condition than in the normoxic condition at sarcomere lengths greater than 2.0 microm. Staining for desmin, the major muscle-type intermediate filament, the longitudinal system of which connects the mitochondria with the Z bands of sarcomeres, showed a clear cross-striation pattern in both papillary muscles with and without the exposure to hypoxia, suggesting that desmin was preserved after the exposure to hypoxia. These data indicate that the mitochondrial length changes according to changes in the sarcomere length, suggesting the possible role of mitochondria as an internal load against myocyte contraction. It is also suggested that mitochondria exposed to hypoxia may be more resistive to both compression and stretch in a longitudinal direction than those in the normoxic condition.