Polly A. Hofmann

C-protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2+ activation

1. Effects on maximum shortening velocity (Vmax) due to partial extraction of C‐protein were investigated in skinned fibres from rabbit psoas muscles. Up to 80% of endogenous C‐protein was extracted, as assessed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of fibre segments obtained before and after the extraction protocol. Vmax was obtained at 15 degrees C by measuring the times required to take up various amounts of slack imposed at one end of the fibre. 2. During maximal activation with Ca2+, Vmax in control fibres was 4.26 +/‐ 0.16 (mean +/‐ S.E.M., n = 7) muscle lengths per second (ML/s). Following extraction of approximately 40% of endogenous C‐protein, Vmax in these same fibres was 4.41 +/‐ 0.24 ML/s. 3. At sufficiently low levels of submaximal activation, high‐ and low‐velocity phases of unloaded shortening were observed. Partial extraction of C‐protein significantly increased Vmax in the low‐velocity phase but had no effect on the high‐velocity phase. The effect on low‐velocity Vmax was fully reversed by re‐addition of purified C‐protein. 4. At low levels of activation, the amount of shortening to the break‐point between the high‐ and low‐velocity phases was not significantly affected by C‐protein extraction. Under control conditions the average break‐point was 85.6 +/‐ 3.1 nm/half‐sarcomere, while 84.1 +/‐ 3.1 nm/half‐sarcomere was obtained following partial extraction of C‐protein. 5. These results are considered in terms of a model in which an internal load slows Vmax at low levels of activation once a given amount of active shortening has occurred. C‐protein may contribute to this internal load either by binding to actin and myosin or by influencing mechanical properties of myosin cross‐bridges.

Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats.

Effects on isometric tension generation and maximum velocity of unloaded shortening after exposure to cAMP-dependent protein kinase (PKA) were investigated in rat enzymatically isolated, tritonized ventricular myocytes. Exposure of myocytes to PKA in the presence of [32P]ATP resulted in phosphorylation of troponin I and C protein. Ca2+ sensitivity of isometric tension was assessed as pCa50, ie, the [Ca2+] at which tension was 50% of maximum, and was lower after PKA treatment (pCa50 5.58) than before PKA treatment (pCa50 5.74). This suggests beta-adrenergic stimulation of the heart and subsequent increases in PKA activity and phosphorylation of troponin I and C protein lead to a significant decrease in tension-generating ability at a given submaximum [Ca2+]. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of the cardiac myocyte preparation. Unloaded shortening velocity during maximum activation was 2.88 +/- 0.11 muscle lengths per second (mean +/- SEM) before PKA exposure and 2.86 +/- 0.13 muscle lengths per second after PKA exposure. Unloaded shortening velocity during 40% of maximum activation was 1.91 +/- 0.25 muscle lengths per second before PKA exposure and 2.17 +/- 0.15 muscle lengths per second after PKA exposure. The absence of an effect of PKA on unloaded shortening velocity in skinned ventricular myocytes suggests that beta-adrenergic stimulation of myocardium either does not affect myofilament velocity of shortening or alters velocity of shortening by a non-PKA-dependent process.

Altered calcium sensitivity of isometric tension in myocyte-sized preparations of porcine postischemic stunned myocardium.

Postischemic ventricular myocardial dysfunction, termed stunning, is characterized by a persistent but ultimately reversible depression of contractile function. The present study was undertaken to investigate the possibilities that reduced contractile force in stunning is due to a decrease in maximal tension-generating capability or to a decrease in the Ca2+ sensitivity of the myofilaments. The experiments combine an in vivo open-chest porcine heart model of stunning (n = 5) with in vitro measures of myocyte myofilament calcium sensitivity from these same hearts. Regional myocardial function in the left anterior descending coronary artery (LAD) perfusion bed of porcine hearts was measured with transmural ultrasonic crystals. The protocol was 45 minutes of low-flow LAD ischemia at 40% of control flow, followed by 30 minutes of postischemic reperfusion at control aerobic flow. Percent systolic wall thickening decreased to 8 +/- 5% of control during ischemia (p < 0.05) and returned to 38 +/- 8% of control in the postischemic stunned state (p < 0.05). Serial endocardial biopsies were obtained from the preischemic and postischemic myocardium in the LAD perfusion bed and from the aerobically perfused myocardium in the circumflex bed. The biopsies were mechanically disrupted, and myocyte-sized preparations of permeabilized myocardium were attached to a force transducer and a length-changing device to allow for direct measurement of steady-state tension-pCa (i.e., -log[Ca2+]) relations. The pCa for half-maximal activation of tension, i.e., pCa50, in LAD myocardium decreased from 5.88 +/- 0.05 before ischemia to 5.69 +/- 0.03 after ischemia (p < 0.05); however, maximal Ca(2+)-activated tension and the slope of the tension-pCa relation were unaffected by the ischemic episode.(ABSTRACT TRUNCATED AT 250 WORDS)

Bound calcium and force development in skinned cardiac muscle bundles: Effect of sarcomere length

There is evidence that the steep ascending limb of the force-length curve in cardiac muscle (Frank-Starling relation) is based on a length-dependence of myofilament Ca2+ sensitivity. Previous work from this laboratory has indicated that in the sarcomere length range corresponding to the ascending limb of the cardiac force length curve (1.7 to 2.3 microns) the Ca2+-troponin C affinity is length-dependent. In this study Ca2+ binding to chemically skinned bovine cardiac muscle bundles was measured during ATP-induced force generation with fiber bundles having sarcomere lengths of 2.2 to 2.4 microns and 1.6 to 1.8 microns. A double isotope technique was used to make concurrent determinations of the force-pCa and bound Ca2+-pCa relationships. At the longer sarcomere lengths the fibers bound, at saturation, an amount of Ca2+ equivalent to approximately 3 mol Ca2+/mol troponin C. Force development appeared to be coupled to titration of the single, low-affinity Ca2+-specific site. In the pCa range 7.0 to 6.0 sarcomere length had no effect on Ca2+ binding. In the pCa range 6.0 to 5.0, in which force increased steeply, there was, in addition to a decreased relative force, a significant reduction in bound Ca2+ at the shorter sarcomere length. Thus sarcomere length appears to influence the Ca2+ binding properties of the regulatory site on troponin C. These data provide direct evidence that length-dependent modulation of Ca2+-troponin C affinity may make a major contribution to the force-length relationship in cardiac muscle.

Effects of Constitutive Overexpression of Insulin-Like Growth Factor-1 on the Mechanical Characteristics and Molecular Properties of Ventricular Myocytes

Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca2+ sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca2+ levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca2+ sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V1 in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.

Effects of calcium on shortening velocity in frog chemically skinned atrial myocytes and in mechanically disrupted ventricular myocardium from rat.

Effects of [Ca2+] on isometric tension and unloaded shortening velocity were characterized in single chemically skinned myocytes from frog atrium and in mechanically disrupted myocardium from rat ventricle. The preparations were attached to a force transducer and piezoelectric translator and were viewed with an inverted microscope to allow continuous monitoring of sarcomere length during mechanical measurements. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of each preparation. Ca2+ sensitivity of isometric tension was assessed as pCa50, i.e., the Ca2+ concentration at which tension was 50% maximal, and was greater for frog atrial myocytes (pCa50 6.17) than for rat ventricular myocytes (pCa50 6.06). This difference in Ca2+ sensitivity may be due to variations in myofibrillar protein isoform composition in the two preparations. Inclusion of caffeine in the activating solutions substantially increased the Ca2+ sensitivity of tension, which may be a manifestation of a direct effect of caffeine on the myofibrillar proteins. Unloaded shortening velocity during maximal activation averaged 4.32 muscle lengths per second in frog atrial myocytes and 4.46 muscle lengths per second in rat ventricular myocytes. When [Ca2+] was reduced, unloaded shortening velocity decreased substantially in both preparations. Possible mechanisms for the effect of Ca2+ on shortening velocity in myocardium include Ca2+ dependence of the rate of ADP dissociation from actomyosin complexes or a shortening-dependent internal load involving structures such as C protein or long-lived myosin cross-bridges.

Protection from Doxorubicin-Induced Cardiomyopathy Using the Modified Anthracycline N-Benzyladriamycin-14-valerate (AD 198)

The anthracycline doxorubicin (Dox) is an effective antitumor agent. However, its use is limited because of its toxicity in the heart. N-Benzyladriamycin-14-valerate (AD 198) is a modified anthracycline with antitumor efficacy similar to that of Dox, but with significantly less cardiotoxicity and potentially cardioprotective elements. In the present study, we investigated the possibility of in vivo protective effects of low-dose AD 198 against Dox-induced cardiomyopathy. To do this, rats were divided into four groups: vehicle, Dox (20 mg/kg; single injection day 1), AD 198 (0.3 mg/kg per injection; injections on days 1, 2, and 3), or a combination treatment of Dox + AD 198. Seventy-two hours after beginning treatment, hearts from the Dox group had decreased phosphorylation of AMP kinase and troponin I and reduced poly(ADP-ribose) polymerase, β-tubulin, and serum albumin expression. Dox also increased the phosphorylation of phospholamban and expression of inducible nitric-oxide synthase in hearts. Each of these Dox-induced molecular changes was attenuated in the Dox + AD 198 group. In addition, excised hearts from rats treated with Dox had a 25% decrease in left ventricular developed pressure (LVDP) and a higher than normal increase in LVDP when perfused with a high extracellular Ca2+ solution. The Dox-induced decrease in baseline LVDP and hyper-responsiveness to [Ca2+] was not observed in hearts from the Dox + AD 198 group. Thus Dox, with well established and efficient antitumor protocols, in combination with low levels of AD 198, to counter anthracycline cardiotoxicity, may be a promising next step in chemotherapy.

Endotoxemia-induced myocardial dysfunction is not associated with changes in myofilament Ca2+ responsiveness

Myocardial contractile function is depressed after onset of endotoxemia and is intrinsic to the ventricular myocyte. We tested the hypothesis that decreased Ca2+ responsiveness of the contractile myofilaments underlies this inotropic depression. Specifically, we evaluated the relationship between Ca2+ and unloaded cell shortening and isometric tension development of skinned guinea pig ventricular myocytes. Myocytes were isolated 4 h after intraperitoneal injection of 4 mg/kg Escherichia colilipopolysaccharide (LPS) or saline (control; Ctl). Myofilament Ca2+ responsiveness assessed by image analysis of shortening of skinned myocytes at pH 7.0 was not different between Ctl [pCa value that resulted in half-maximal shortening (pCa50): 5.78 ± 0.04] and LPS (pCa50: 5.72 ± 0.02). Similarly, myofilament Ca2+ responsiveness measured by isometric tension of skinned myocytes was not different between Ctl (pCa50: 5.73 ± 0.02) and LPS (pCa50: 5.76 ± 0.02). Maximal tension generated by LPS myocytes (2.89 ± 0.23 g/mm2) was significantly less ( P < 0.05) than Ctl (3.75 ± 0.34 g/mm2). However, when myocytes were isolated and skinned in the presence of protease inhibitors, maximal tension generated by LPS myocytes (3.53 ± 0.98 g/mm2) was similar to Ctl (3.01 ± 0.80 g/mm2). We conclude that in vivo administration of LPS resulting in endotoxemia without shock does not alter myofilament Ca2+ responsiveness of ventricular myocytes. Rather, reduced contractility is more likely a result of decreased Ca2+ availability because systolic Ca2+ transients of fura 2-loaded LPS myocytes were significantly decreased ( P < 0.05) compared with Ctl myocytes.

A cardioprotective role for platelet-activating factor through NOS-dependent S-nitrosylation

Controversy exists as to whether platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, can actually protect the heart from postischemic injury. To determine whether endogenous activation of the PAF receptor is cardioprotective, we examined postischemic functional recovery in isolated hearts from wild-type and PAF receptor-knockout mice. Postischemic function was reduced in hearts with targeted deletion of the PAF receptor and in wild-type hearts treated with a PAF receptor antagonist. Furthermore, perfusion with picomolar concentrations of PAF improved postischemic function in hearts from wild-type mice. To elucidate the mechanism of a PAF-mediated cardioprotective effect, we employed a model of intracellular Ca2+ overload and loss of function in nonischemic ventricular myocytes. We found that PAF receptor activation attenuates the time-dependent loss of shortening and increases in intracellular Ca2+ transients in Ca2+ -overloaded myocytes. These protective effects of PAF depend on nitric oxide, but not activation of cGMP. In addition, we found that reversible S-nitrosylation of myocardial proteins must occur in order for PAF to moderate Ca2+ overload and loss of myocyte function. Thus our data are consistent with the hypothesis that low-level PAF receptor activation initiates nitric oxide-induced S-nitrosylation of Ca2+ -handling proteins, e.g., L-type Ca2+ channels, to attenuate Ca2+ overload during ischemia-reperfusion in the heart. Since inhibition of the PAF protective pathway reduces myocardial postischemic function, our results raise concern that clinical therapies for inflammatory diseases that lead to complete blockade of the PAF receptor may eliminate a significant, endogenous cardioprotective pathway.

N-Benzyladriamycin-14-valerate (AD 198): a non-cardiotoxic anthracycline that is cardioprotective through PKC-epsilon activation.

N-Benzyladriamycin-14-valerate (AD 198) is one of several novel anthracycline protein kinase C (PKC)-activating agents developed in our laboratories that demonstrates cytotoxic superiority over doxorubicin (Adriamycin; DOX) through its circumvention of multiple mechanisms of drug resistance. This characteristic is attributed at least partly to the principal cellular action of AD 198: PKC activation through binding to the C1b (diacylglycerol binding) regulatory domain. A significant dose-limiting effect of DOX is chronic, dose-dependent, and often irreversible cardiotoxicity ascribed to the generation of reactive oxygen species (ROS) from the semiquinone ring structure of DOX. Despite the incorporation of the same ring structure in AD 198, we hypothesized that AD 198 might also be cardioprotective through its ability to activate PKC-ϵ, a key component of protective ischemic preconditioning in cardiomyocytes. Chronic administration of fractional LD50 doses of DOX and AD 198 to mice results in histological evidence of dose-dependent ventricular damage by DOX but is largely absent from AD 198-treated mice. The absence of significant cardiotoxicity with AD 198 occurs despite the equal ability of DOX and AD 198 to generate ROS in primary mouse cardiomyocytes. Excised rodent hearts perfused with AD 198 prior to hypoxia induced by vascular occlusion are protected from functional impairment to an extent comparable to preconditioning ischemia. AD 198-mediated cardioprotection correlates with increased PKC-ϵ activation and is inhibited in hearts from PKC-ϵ knockout mice. These results suggest that, despite ROS production, the net cardiac effect of AD 198 is protection through activation of PKC-ϵ.