Matthew R. Wolff

Reduction in density of transverse tubules and L-type Ca2+ channels in canine tachycardia-induced heart failure

OBJECTIVE Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation-contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca(2+) through L-type Ca(2+) channels (I(Ca)) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca(2+) channels change in canine tachycardia pacing-induced cardiomyopathy. METHODS Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I(Ca) and intramembrane charge movement. RESULTS A complex staining pattern of interconnected tubules including prominent transverse components spaced every approximately 1.6 microm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5+/-0.4 in control to 8.7+/-0.4% in failing cells (P<0.001). Whole-cell patch clamp studies revealed that I(Ca) density was unchanged. Since whole-cell I(Ca) is due to both the number of channels as well as the functional properties of those channels, we measured intramembrane charge movement as an assay for changes in channel number. The saturating amount of charge that moves due to gating of L-type Ca(2+) channels, Q(on,max), was decreased from 6.5+/-0.6 in control to 2.8+/-0.3 fC/pF in failing myocytes (P<0.001). CONCLUSIONS Cellular remodeling in heart failure results in decreased density of T-tubules and L-type Ca(2+) channels, which contribute to abnormal EC coupling.

Abnormal Ca2+ Release, but Normal Ryanodine Receptors, in Canine and Human Heart Failure

Abstract— Sarcoplasmic reticulum (SR) Ca2+ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca2+ transport. Intracellular Ca2+ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca2+]i transient were reduced in HF. Ca2+ uptake assays showed 44±8% reduction of Vmax in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30±5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The Po of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor &bgr;-adrenergic stimulation of Ca2+ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca2+ uptake, more than Ca2+ release, contributing to the depressed and slow Ca2+ transient characteristic of HF.

Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation.

To examine the role of alterations in myofibrillar function in human dilated cardiomyopathies, we determined isometric tension-calcium relations in permeabilized myocytesized myofibrillar preparations (n = 16) obtained from left ventricular biopsies from nine patients with dilated cardiomyopathy (DCM) during cardiac transplantation or left ventricular assist device implantation. Similar preparations (n = 10) were obtained from six normal hearts used for cardiac transplantation. Passive and maximal Ca2+-activated tensions were similar for the two groups. However, the calcium sensitivity of isometric tension was increased in DCM compared to nonfailing preparations ([Ca2+]50=2.46+/-0.49 microM vs 3.24+/-0.51 microM, P < 0.001). In vitro treatment with the catalytic subunit of protein kinase A (PKA) decreased calcium sensitivity of tension to a greater degree in failing than in normal preparations. Further, isometric tension-calcium relations in failing and normal myofibrillar preparations were similar after PKA treatment. These findings suggest that the increased calcium sensitivity of isometric tension in DCM may be due at least in part to a reduction of the beta-adrenergically mediated (PKA-dependent) phosphorylation of myofibrillar regulatory proteins such as troponin I and/or C-protein.

Rate of Tension Development in Cardiac Muscle Varies With Level of Activator Calcium

In skeletal muscle, the rate of transition from weakly bound to force-generating crossbridge states increases as calcium concentration is increased. To examine possible calcium sensitivity of this transition in cardiac muscle, we determined the kinetics of isometric tension development during steady activation in detergent-permeabilized rat ventricular trabeculae (n = 7) over a range of calcium concentrations. Force-generating crossbridges in activated trabeculae were disrupted by a brief, rapid release and restretch equivalent to 20% muscle length (15 degrees C), which resulted in a subsequent phase of tension redevelopment that was well fit by a monoexponential function (rate constant, ktr). Sarcomere length was monitored by laser diffraction and held constant during tension redevelopment by an iterative adaptive feedback control system. The ktr increased from 3.6 +/- 0.8 s-1 at the lowest calcium concentration studied (pCa 5.9) to 9.5 +/- 1.3 s-1 during maximal activation (pCa 4.5). The relationship between relative ktr and relative tension was approximately linear over a wide range of [Ca2+] (r2 = .94). This result differs quantitatively from results in skeletal muscle, in which ktr is sensitive to [Ca2+] primarily at higher activation levels. This observation is also inconsistent with a recent suggestion that the rate of force development in living myocardium is independent of the activation level. Our results in skinned myocardium can be explained by a model in which calcium is a graded regulator of both the extent and rate of binding of force-generating crossbridges to the thin filament.

Calcium Sensitivity of Isometric Tension Is Increased in Canine Experimental Heart Failure

To examine the role of alterations in myofibrillar function in chronic heart failure, we determined isometric tension-pCa relations in permeabilized myocardium from a canine model of dilated cardiomyopathy (DCM) produced by chronic rapid pacing. In the initial series of experiments, seven dogs were paced at 250 beats per minute for 28.9 +/- 7.0 days, resulting in ventricular dilatation and reduced ejection fractions by echocardiography and elevated intracardiac filling pressures. Isometric tension-pCa relations were measured by using mechanically disrupted and permeabilized myocyte-sized preparations obtained from left ventricular biopsies before (n = 11) and after (n = 10) chronic rapid pacing-induced heart failure. Resting sarcomere length (SL) was set at 2.35 microns, and preparations had low end compliance (SL was 2.23 +/- 0.03 microns during maximal activation). Passive tension (2.1 +/- 1.0 versus 2.4 +/- 0.6 mN/mm2) and maximal Ca(2+)-activated tension (25.9 +/- 9.3 versus 27.8 +/- 6.8 mN/mm2) were similar for control and DCM preparations, respectively. However, the calcium sensitivity of isometric tension was increased in failing myocardium (pCa50 5.95 +/- 0.11 [DCM] versus 5.83 +/- 0.10 [control], P = .001). Treatment of myofibrillar preparations with the catalytic subunit of protein kinase A decreased calcium sensitivity of tension to a greater degree in failing preparations (shift of pCa50 from 6.04 +/- 0.06 to 5.75 +/- 0.09, n = 7) than in nonfailing preparations (5.91 +/- 0.08 to 5.74 +/- 0.07, n = 8), and isometric tension-pCa relations in the two groups were not significantly different after protein kinase A treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Sarcomere length dependence of the rate of tension redevelopment and submaximal tension in rat and rabbit skinned skeletal muscle fibres

1 We examined the hypothesis that in skeletal muscle the steep relationship between twitch tension and sarcomere length (SL) within the range 2.30 to 1.85 μm involves SL‐dependent alterations in the rate of tension development. 2 In skinned preparations of both rat slow‐twitch and rabbit fast‐twitch skeletal muscle fibres the rate of tension redevelopment (ktr) at 15°C was reduced at short SL (∼2.00/μm) compared with a longer SL (∼2.30 μm). In submaximally activated fibres, the decrease in ktr over this range of lengths was greater in fast‐twitch fibres (38% reduction) than in slow‐twitch fibres (14% reduction). 3 Ca2+ sensitivity of tension, as assessed as the pCa (−log[Ca2+]) for half‐maximal activation, or pCa50, decreased to a greater extent in rabbit fast‐twitch skeletal muscle fibres than in slow‐twitch fibres from both rabbit and rat when SL was reduced from ∼2.30 to ∼1.85 μm. The ΔpCa50 over this SL range was 0.24 ± 0.07 pCa units in fast‐twitch fibres from rabbit psoas muscle. The ΔpCa50 for slow‐twitch fibres from rabbit and rat soleus muscle was 0.08 ± 0.02 and 0.10 ± 0.04 pCa units, respectively. 4 Osmotic compression of both slow‐twitch and fast‐twitch fibres at a SL of 2.00 μm increased ktr to values similar to those obtained at a SL of 2.30 μm in the absence of dextran. This result indicates that the slower rate of tension redevelopment at short SL is due in large part to the increase in interfilament lattice spacing associated with shorter SL. 5 Taken together, these results suggest that length dependence of twitch tension is, in part, due to length dependence of isometric cross‐bridge interaction kinetics, an effect that is mediated by length‐dependent changes in interfilament lattice spacing.

Force‐velocity and power‐load curves in rat skinned cardiac myocytes

1 This study utilized a skinned myocyte preparation with low end compliance to examine force‐velocity and power‐load curves at 12 °C in myocytes from rat hearts. 2 In maximally activated myocyte preparations, shortening velocities appeared to remain constant during load clamps in which shortening took place over a sarcomere length range of ≈2.30‐2.00 μm. These results suggest that previously reported curvilinear length traces during load clamps of multicellular preparations were due in part to extracellular viscoelastic structures that give rise to restoring forces during myocardial shortening. 3 During submaximal Ca2+ activations, the velocity of shortening at low loads slowed and the time course of shortening became curvilinear, i.e. velocity progressively slowed as shortening continued. This result implies that cross‐bridge cycling kinetics are slower at low levels of activation and that an internal load arises during shortening of submaximally activated myocytes, perhaps due to slowly detaching cross‐bridges. 4 Reduced levels of activator Ca2+ also reduced maximal power output and increased the relative load at which power output was optimal. For a given absolute load, the shift has the effect of maintaining power output near the optimum level despite reductions in cross‐bridge number and force generating capability at lower levels of Ca2+.

Ca2+ Binding to Troponin C in Skinned Skeletal Muscle Fibers Assessed with Caged Ca2+ and a Ca2+ Fluorophore INVARIANCE OF Ca2+ BINDING AS A FUNCTION OF SARCOMERE LENGTH

Ca2+ sensitivity of tension varies with sarcomere length in both skeletal and cardiac muscles. One possible explanation for this effect is that the Ca2+ affinity of the regulatory protein troponin C decreases when sarcomere length is reduced. To examine length dependence of Ca2+ binding to troponin C in skeletal muscle, we developed a protocol to simultaneously monitor changes in sarcomere length, tension, and Ca2+ concentration following flash photolysis of caged Ca2+. In this protocol, [Ca2+] was rapidly increased by flash photolysis of caged Ca2+, and changes in [Ca2+] due to photolysis and the subsequent binding to troponin C were assessed using a Ca2+ fluorophore. Small bundles of fibers from rabbit skinned psoas muscles were loaded with Ca2+ fluorophore (Fluo-3) and caged Ca2+ (dimethoxynitrophenamine or o-nitrophenyl-EGTA). The bundles were then transferred to silicone oil, where [Ca2+]free, tension, and sarcomere length were monitored before and after photolysis of caged Ca2+. Upon photolysis of caged Ca2+, fluorescence increased and then decayed to a new steady-state level within ∼1 s, while tension increased to a new steady-state level within ∼1.5 s. After extracting troponin C, fibers did not generate tension following the flash, but steady-state post-flash fluorescence was significantly greater than when troponin C was present. The difference in [Ca2+]free represents the amount of Ca2+ bound to troponin C. In fibers that were troponin C-replete, Ca2+ binding to troponin C did not differ at short (∼1.97 μm) and long (∼2.51 μm) sarcomere length, yet tension was ∼50% greater at the long sarcomere length. These results show that the affinity of troponin C for Ca2+ is not altered by changes in sarcomere length, indicating that length-dependent changes in Ca2+ sensitivity of tension in skeletal muscle are not related to length-dependent changes in Ca2+ binding affinity of troponin C.

Left Atrial Volume Is Associated with Inflammation and Atherosclerosis in Patients with Kidney Disease

Background: Left atrial volume (LAV) is an independent echocardiographic predictor of cardiovascular events in the general population. We evaluated predictors of LAV in patients with advanced chronic kidney disease (CKD). Hypothesis: Increasing LAV identifies increased cardiovascular risk in patients with CKD. Methods: Transthoracic echocardiography was performed in CKD patients undergoing cardiovascular evaluation prior to listing for renal transplantation. LAV was measured using the biplane area‐length formula and indexed for body surface area. Carotid intima‐media thickness was assessed by B‐mode ultrasound. Lipoproteins were measured by nuclear magnetic resonance spectroscopy. Values are presented as mean (standard deviation). Relationships with LAV were evaluated using univariate and multivariable regression analyses. Results: There were 99 participants (80% white, 68% male). Their mean age was 55.7 (9.3) years. Significant correlates of LAV were systolic blood pressure (r = 0.24), C‐reactive protein (r = 0.29), carotid intima‐media thickness (r = 0.29), peak transmitral E‐wave (r = 0.38), and severity of mitral regurgitation (r = 0.23, P < 0.05 for all). LAV also was higher among individuals with a history of stroke (45 mL/m2 vs 36.3 mL/m2, P = 0.04) and with >75% stenosis on coronary angiography (38.4 mL/m2 vs 31.8 mL/m2, P = 0.03). In regression models, high sensitivity CRP (hs‐CRP), the transmitral E‐wave velocity, and a history of stroke independently predicted LAV (P ≤ 0.05). Conclusion: In individuals with advanced CKD, LAV is associated with inflammation, increased early transmitral filling velocities, and atherosclerosis. These findings may indicate increased cardiovascular risk with increasing LAV in patients with CKD.

Mitral Annular Calcification is Associated with Reduced Left Ventricular Function and Inflammation in Patients with Chronic Kidney Disease

BACKGROUND Mitral annular calcification (MAC) is prevalent in patients with chronic kidney disease (CKD); however, it is not known whether the increased cardiovascular risk observed in patients with CKD and MAC is related to atherosclerotic burden, because they share common risk factors. METHODS Transthoracic echocardiography was performed in patients with CKD undergoing pre-kidney transplantation evaluation. Fasting lipids, high-sensitivity C-reactive protein, parathyroid hormone, calcium, and creatinine levels were measured. RESULTS Of 99 participants, the 31 with MAC had higher carotid intima-media thickness (P = .004), lower left ventricular ejection fraction (P = .016), and higher high-sensitivity C-reactive protein (P = .01). MAC was predicted independently by increasing high-sensitivity C-reactive protein, decreasing left ventricular ejection fraction, and not being on dialysis (likelihood ratio 21.8, P < .001). Models were not affected significantly by the addition of age, carotid intima-media thickness, and other laboratory tests. CONCLUSIONS In patients with CKD, MAC is associated with inflammation, reduced left ventricular function, and treatment with dialysis, independent of the degree of subclinical atherosclerosis.