Cardiomyocyte Relaxation Research Articles

Skeletal myoblasts and bone marrow cells enhance contractility of failing cardiomyocytes in co-culture

Cell transplantation has been shown to enhance ventricular function in heart failure, but the mechanisms responsible remain unknown. We hypothesized that bone marrow mononuclear cells and skeletal myoblasts cells can influence cardiomyocytes by paracrine mechanisms. Myocardial infarction was induced in adult female Sprague–Dawley rats. Left ventricular ejection fraction after 3 weeks was 35.5±2.0%. Isolated failing left ventricular myocytes were cultured (density 5.2 cells/mm2) on their own (Control) or with either bone marrow mononuclear cells (BM, density 52 cells/mm2) or skeletal myoblasts (SK, density 5.2 cells/mm2). BM and SK were separated from the failing cardiomyocytes by a Transwell™ porous membrane. After 48 h of co-culture with SK or BM cardiomyocyte relaxation speed was increased. The decay time course of the indo-1 transient was also hastened by SK. SK, but not BM, also increased the cardiomyocyte contraction amplitude compared to Control. Tabled 1 Control BM SK Sarcomere shortening (nm) 46±6 55±1 91±14* Sarcomere relaxation T50 (ms) 34±6 18±3* 13±2* Indo-1 decay T50 (ms) 22±2 18±2 13±1* (Data represented as mean±SEM, *p<0.05 vs. Control) Open table in a new tab

Cardiac overexpression of antioxidant catalase attenuates aging-induced cardiomyocyte relaxation dysfunction

Catalase, an enzyme which detoxifies H 2O 2, may interfere with cardiac aging. To test this hypothesis, contractile and intracellular Ca 2+ properties were evaluated in cardiomyocytes from young (3–4 months) and old (26–28 months) FVB and transgenic mice with cardiac overexpression of catalase. Contractile indices analyzed included peak shortening (PS), time-to-90% PS (TPS 90), time-to-90% relengthening (TR 90), half-width duration (HWD), maximal velocity of shortening/relengthening (±d L/d t) and intracellular Ca 2+ levels or decay rate. Levels of advanced glycation endproduct (AGE), Na +/Ca 2+ exchanger (NCX), sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA2a), phospholamban (PLB), myosin heavy chain (MHC), membrane Ca 2+ and K + channels were measured by western blot. Catalase transgene prolonged survival while did not alter myocyte function by itself. Aging depressed ±d L/d t, prolonged HWD, TR 90 and intracellular Ca 2+ decay without affecting other indices in FVB myocytes. Aged FVB myocytes exhibited a stepper decline in PS in response to elevated stimulus or a dampened rise in PS in response to elevated extracellular Ca 2+ levels. Interestingly, aging-induced defects were nullified or significantly attenuated by catalase. AGE level was elevated by 5-fold in aged FVB compared with young FVB mice, which was reduced by catalase. Expression of SERCA2a, NCX and Kv 1.2 K + channel was significantly reduced although levels of PLB, L-type Ca 2+ channel dihydropyridine receptor and β-MHC isozyme remained unchanged in aged FVB hearts. Catalase restored NCX and Kv 1.2 K + channel but not SERCA2a level in aged mice. In summary, our data suggested that catalase protects cardiomyocytes from aging-induced contractile defect possibly via improved intracellular Ca 2+ handling.

Abstract 580: Skeletal Myoblasts and Bone Marrow-Derived Cells Enhance the Contractile Function of Isolated Failing Cardiomyocytes in Co-culture

Cell transplantation has been shown to enhance ventricular function in heart failure. However, the low numbers of surviving transplanted cells can not explain the functional myocardial improvement directly, and the cellular mechanisms responsible remain unknown. We hypothesized that transplanted cells influence the native myocardium by paracrine mechanisms, and investigated the in vitro effects of bone marrow mononuclear cells or skeletal myoblasts on neighboring cardiomyocytes isolated from a rat model of heart failure. Myocardial infarction was induced in adult female Sprague-Dawley rats by left coronary artery ligation. Left ventricular ejection fraction after 3 weeks was 35.5 ± 2.0%. Isolated left ventricular myocytes were cultured (density 5.2 cells/mm 2 ) on their own (Control) or with either bone marrow mononuclear cells (BM, density 52 cells/mm 2 ) or skeletal myoblasts (SK, density 5.2 cells/mm 2 ). After 48 hours of culture with SK or BM cardiomyocyte relaxation speed was increased (time to 50% relaxation, in ms - Control 34 ± 6 [ 28 ]; BM 18 ± 3 [ 16 ] p &lt; 0.05 vs Control; SK 13 ± 2 [ 20 ] p &lt; 0.05 vs Control). The decay time course of the Ca 2+ transient, assessed by indo-1 fluorescence, was also hastened by SK (time to 50% decay, in ms – HF 22 ± 2 [ 29 ]; BM 18 ± 2 [ 17 ] ns ; SK 13 ± 1 [ 17 ] p &lt; 0.05 vs Control). SK also increased the contraction amplitude compared to Control (sarcomere shortening, in μm - HF 0.046 ± 0.006 [ 28 ]; BM 0.055 ± 0.01 [ 16 ] ns ; SK 0.091 ± 0.014 [ 20 ] p &lt; 0.05 vs Control). We conclude that bone marrow mononuclear cells and skeletal myoblasts enhance cardiomyocyte function in co-culture, demonstrating that paracrine mechanisms are involved. This may account for the functional improvement seen after cell transplantation.

New Molecular Mechanism in Diastolic Heart Failure

In contrast to systolic heart failure (SHF), for which knowledge of pathophysiology and therapy has advanced rapidly over the past decade, little is known about diastolic heart failure (DHF). The article by van Heerebeek et al1 in this issue of Circulation that describes an abnormal distribution of titin isoforms in DHF may herald a new approach to understanding the pathophysiology of this syndrome. Article p 1966 Recognition of 2 forms of heart failure is not new; almost 70 years ago, Fishberg2 described “those forms of cardiac insufficiency which are due to inadequate diastolic filling of the heart (hypodiastolic failure) [and] the far more common ones in which the heart fills adequately but does not empty to the normal extent (hyposystolic failure)” (p 23). This distinction has stood the test of time, because there is a growing consensus that these 2 clinical syndromes differ in epidemiology, demographics, and origin. Because DHF and SHF represent subgroups of patients with heart failure, they share many clinical features, notably the hemodynamic findings, but it is now clear that they are caused by different pathophysiological mechanisms. Hearts in SHF are characterized by eccentric hypertrophy, progressive left ventricular (LV) dilation, and abnormal LV systolic properties, whereas in DHF, the hearts generally exhibit concentric hypertrophy, normal or reduced LV volume, concentric remodeling, and abnormal diastolic function.3,4 In addition, cardiomyocyte size, shape, and molecular composition differ in these 2 syndromes. Diastolic dysfunction refers to mechanical and functional abnormalities present during relaxation and filling, whereas DHF refers to clinical syndromes in which patients with heart failure have little or no ventricular dilatation and significant, often dominant diastolic dysfunction. Diastolic dysfunction can be quantified with indices of LV pressure decline and filling. Abnormal pressure decline is characterized by decreased peak −dP/dt, prolonged isovolumic time constant (τ), and …

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9–12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42–63%) compared to starch (ST)-fed controls. Cytosolic Ca 2+ removal attributable to Na +/Ca 2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca 2+ and cytosolic Ca 2+ clearing through NCX were normal, whereas Ca 2+ uptake by SERCA was significantly slower in SU myocytes (330 ± 29 ms) compared to ST cells (253 ± 16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca 2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca 2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).

Gene transfer of parvalbumin improves diastolic dysfunction in senescent myocytes.

Impaired relaxation is a cardinal feature of senescent myocardial dysfunction. Recently, adenoviral gene transfer of parvalbumin, a small calcium-buffering protein found exclusively in skeletal muscle and neurons, has been shown to improve cardiomyocyte relaxation in disease models of diastolic dysfunction. The goal of this study was to investigate whether parvalbumin gene transfer could reverse diastolic dysfunction in senescent cardiomyocytes. Myocytes were isolated from senescent (26 months) and adult (6 months) F344/BN hybrid rats and were infected with Ad.Parv.GFP (where GFP is green fluorescent protein) or Ad.betagal.GFP at a multiplicity of infection of 250 for 48 hours. Uninfected senescent and adult myocytes served as controls. After stimulation at a frequency of 0.5 Hz, intracellular calcium transients and myocyte contractility were measured using dual excitation spectrofluorometry and video-edge detection system (Ionoptix). Parvalbumin significantly improved relaxation parameters in senescent myocytes: Both the rate of calcium transient decay and the rate of myocyte relengthening were dramatically increased in senescent cardiac myocytes transduced with parvalbumin compared with nontransduced and GFP-expressing controls, with no effect on myocyte shortening. Parvalbumin expression corrects impaired relaxation in aging myocytes. Given that abnormalities of myocyte relaxation underlie diastolic dysfunction in a large proportion of elderly patients with heart failure, gene transfer of parvalbumin may thus be a novel approach to target diastolic dysfunction in senescent myocardium.

Functional genomics of pressure-loaded cardiomyocytes: etomoxir in heart failure?

Drugs for counteracting the neuroendocrine activation in heart failure can reduce the adverse remodelling of the extracellular matrix of the heart. Progression of heart failure can, however, often not be prevented and the question arises whether important pharmacological targets remain unidentified. Promising are drugs targeted at ventricular diastolic dysfunction which is a marker of early progression of heart failure. Left ventricular dysfunction is characteristic of overloaded hypertrophied hearts with molecular structures that are not adapted to the increased Ca2+ diffusion distances. Thus, the Ca(2+)-pump (SERCA2) of sarcoplasmic reticulum is inadequately expressed leading to a reduced force development and relaxation of hypertrophied cardiomyocytes. Drugs in development (CPT-1 inhibitor/PPARalpha activator) that increase glucose oxidation can enhance SERCA2 expression. The lead compound etomoxir had a selective influence on the contraction and relaxation rate of pressure-overloaded hearts. The functional parameters were correlated with the proportion of alpha-myosin heavy chains. Since viral or inflammatory injury of the heart can also induce a fetal phenotype, metabolic modulators such as etomoxir represent a promising therapeutic approach also for cardiomyopathies with inadequate SERCA2 expression.

Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart

1. To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene-encoded ssTnI. 2. The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3. Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP-dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild-type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non-transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4. The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild-type myocytes. However, the half-time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re-lengthening time of the transgenic cells. 5. These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6. Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to beta-adrenergic receptor stimulation.

Pentameric Assembly of Phospholamban Facilitates Inhibition of Cardiac Function in Vivo

Phospholamban has been proposed to coexist as pentamers and monomers in native sarcoplasmic reticulum membranes. To determine its functional unit in vivo, we reintroduced wild-type (pentameric) or monomeric mutant (C41F) phospholamban in the hearts of phospholamban knockout mice. Transgenic lines, expressing similar levels of mutant or wild-type phospholamban, were identified, and their cardiac phenotypes were characterized in parallel. Sarcoplasmic reticulum Ca2+ transport assays indicated similar decreases in SERCA2 Ca2+ affinity by mutant or wild-type phospholamban. However, the time constants of relaxation and Ca2+ transient decline in isolated cardiomyocytes were diminished to a greater extent by wild-type than mutant phospholamban, even without significant differences in the amplitudes of myocyte contraction and Ca2+ transients between the two groups. Langendorff perfusion also indicated that mutant phospholamban was not capable of depressing the enhanced relaxation parameters of the phospholamban knockout hearts to the same extent as wild-type phospholamban. Moreover, in vivo assessment of mouse hemodynamics revealed a greater depression of cardiac function in wild-type than mutant phospholamban hearts. Thus, the mutant or monomeric form of phospholamban was not as effective in slowing Ca2+ decline or relaxation in cardiomyocytes, hearts, or intact animals as wild-type or pentameric phospholamban. These findings suggest that pentameric assembly of phospholamban is necessary for optimal regulation of myocardial contractility in vivo.

Mechanisms of Cardiomyocyte Dysfunction in Heart Failure Following Myocardial Infarction in Rats

Available information regarding the cellular and molecular mechanisms for reduced myocardial function after myocardial infarction (MI) is scarce. In rats with congestive heart failure (CHF), we examined cardiomyocytes isolated from the non-infarcted region of the left ventricle 6 weeks after ligation of the left coronary artery. Systolic left-ventricular pressure was reduced and diastolic pressure was markedly increased in the CHF-rats. The cardiomyocytes isolated from the CHF-hearts had increased resting length, reduced fractional shortening by 31% and a 34% increase in time to 90% relaxation compared to sham cells (P<0.01 for all). Peakl-type calcium currents were not significantly changed, but peak calcium transients measured with fura-2 were reduced by 19% (P<0.01). Moreover, the decline of the calcium transients as measured by the time constant of a monoexponential function was significantly increased by 26% (P<0.01). We also examined the contribution of the Ca2+-ATPase of the sarcoplasmic reticulum (SR) in the removal of cytosolic Ca2+during relaxation by superfusing cells with 1μmthapsigargin that effectively inhibits the Ca2+-ATPase. Relaxation time in CHF-cells was significantly less prolonged when this drug was used (P<0.01). This suggests that other mechanisms, probably the Na+-Ca2+exchanger, contribute significantly to the relaxation rate in CHF. Simultaneous measurements of fura-2 transients and mechanical shortening did not reveal any alteration in the calcium-myofilament sensitivity in CHF. Our study clearly shows reduced shortening and prolonged relaxation in cardiomyocytes isolated from non-infarcted region of the left ventricle in heart failure. Moreover, we were able to relate the observed cardiomyocyte dysfunction to changes in specific steps in the excitation-contraction coupling.

Relationship between action potential, contraction-relaxation pattern, and intracellular Ca2+ transient in cardiomyocytes of dogs with chronic heart failure.

Abnormalities of contractile function have been identified in cardiomyocytes isolated from failed human hearts and from hearts of animals with experimentally induced heart failure (HF). The mechanism(s) responsible for these functional abnormalities are not fully understood. In the present study, we examined the relationship between action potential duration, pattern of contraction and relaxation, and associated intracellular Ca2+ transients in single cardiomyocytes isolated from the left ventricle (LV) of dogs (n = 7) with HF produced by multiple sequential intracoronary microembolizations. Comparisons were made with LV cardiomyocytes isolated from normal dogs. Action potentials were measured in isolated LV cardiomyocytes by perforated patch clamp, Ca2+ transients by fluo 3 probe fluorescence, and cardiomyocyte contraction and relaxation by edge movement detector. HF cardiomyocytes exhibited an abnormal pattern of contraction and relaxation characterized by an attenuated initial twitch (spike) followed by a sustained contracture ('dome') of 1 to 8 s in duration and subsequent delayed relaxation. This pattern was more prominent at low stimulation rates (58% at 0.2 Hz, n = 211, 21% at 0.5 Hz, n = 185). Measurements of Ca2+ transients in HF cardiomyocytes at 0.2 Hz manifested a similar spike and dome configuration. The dome phase of both the contraction/relaxation pattern and Ca2+ transients seen in HF cardiomyocytes coincided with a sustained plateau of the action potential. Shortening of the action potential duration by administration of saxitoxin (100 nM) or lidocaine (30 microM) reduced the duration of the dome phase of both the contraction/relaxation profile as well as that of the Ca2+ transient profile. An increase of stimulation rate up to 1 Hz caused shortening of the action potential and disappearance of the spike-dome profile in the majority of HF cardiomyocytes. In HF cardiomyocytes, the action potential and Ca2+ transient duration were not significantly different from those measured in normal cells. However, the contraction-relaxation cycle was significantly longer in HF cells (314 +/- 67 ms, n = 21, vs. 221 +/- 38 ms, n = 46, mean +/- SD), indicating impaired excitation-contraction uncoupling in HF cardiomyocytes. The results show that, in cardiomyocytes isolated from dogs with HF, contractile abnormalities and abnormalities of intracellular Ca2+ transients at low stimulation rates are characterized by a spike-dome configuration. This abnormal pattern appears to result from prolongation of the action potential.