Comparative analysis of parvalbumin and SERCA2a cardiac myocyte gene transfer in a large animal model of diastolic dysfunction

Diastolic dysfunction results from impaired ventricular relaxation and is an important component of human heart failure. Genetic modification of intracellular calcium-handling proteins may hold promise to redress diastolic dysfunction; however, it is unclear whether other important aspects of myocyte function would be compromised by this approach. Accordingly, a large animal model of humanlike diastolic dysfunction was established through 1 yr of left ventricular (LV) pressure overload by descending thoracic aortic coarctation in canines. Serial echocardiography documented a progressive increase in LV mass. Diastolic dysfunction with preserved systolic function was evident at the whole organ and myocyte levels in this model, as determined by hemispheric sonomicrometric piezoelectric crystals, pressure transducer catheterization, and isolated myocyte studies. Gene transfer of the sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a) and parvalbumin (Parv), a fast-twitch skeletal muscle Ca(2+) buffer, restored cardiac myocyte relaxation in a dose-dependent manner under baseline conditions. At high Parv concentrations, sarcomere shortening was depressed. In contrast, during beta-adrenergic stimulation, the expected enhancement of myocyte contraction (inotropy) was abrogated by SERCA2a but not by Parv. The mechanism of this effect is unknown, but it could relate to the uncoupling of SERCA2a/phospholamban in SERCA2a myocytes. Considering that inotropy is vital to overall cardiac performance, the divergent effects of SERCA2a and Parv reported here could impact potential therapeutic strategies for human heart failure.     

Genetic manipulation of calcium-handling proteins in cardiac myocytes. II. Mathematical modeling studies

We developed a mathematical model specific to rat ventricular myocytes that includes electrophysiological representation, ionic homeostasis, force production, and sarcomere movement. We used this model to interpret, analyze, and compare two genetic manipulations that have been shown to increase myocyte relaxation rates, parvalbumin (Parv) de novo expression, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) overexpression. The model was used to seek mechanistic insights into 1) the relative contribution of two mechanisms by which SERCA2a overexpression modifies Ca2+ sequestration, i.e., more pumps and an increase in the SERCA2a-to-phospholamban ratio, 2) the mechanisms behind postrest potentiation and how Parv and SERCA2a influence this response, and 3) why Parv myocytes retain their fast kinetics when endogenous SERCA2a is partially impaired by thapsigargin (a condition used to mimic diastolic dysfunction). The model was also utilized to predict whether Parv metal-binding characteristics might be modified to improve diastolic and systolic functions and whether Parv or SERCA2a might affect diastolic Ca2+ levels and myocyte energetics. One outcome of the model was to demonstrate a higher peak and total ATP consumption in SERCA2a myocytes and more even distribution of ATP throughout the cardiac cycle in Parv myocytes. This may have implications for failing hearts that are energetically compromised.     

Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies

Two genetic experimental approaches, de novo expression of parvalbumin (Parv) and overexpression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), have been shown to increase relaxation rates in myocardial tissue. However, the relative effect of Parv and SERCA2a on systolic function and on beta-adrenergic responsiveness at varied pacing rates is unknown. We used gene transfer in isolated rat adult cardiac myocytes to gain a fuller understanding of Parv/SERCA2a function. As demonstrated previously, when Parv is expressed in elevated concentration (>0.1 mM), the transduced myocytes showed a reduction in sarcomere-shortening amplitude: 129 +/- 17, 81 +/- 8, and 149 +/- 14 nm for control, Parv, and SERCA2a, respectively. At physiological temperature, shortening amplitude responses of Parv and SERCA2a myocytes to the beta-adrenergic agonist isoproterenol (Iso) were not statistically different from that of control myocytes. However, in SERCA2a myocytes, in which baseline was slightly elevated and the Iso-stimulated value was slightly lower, the increase in shortening was slightly less than in Parv or control myocytes: 108 +/- 14, 169 +/- 39, and 34 +/- 12% for control, Parv, and SERCA2a, respectively. In another test set, Parv myocytes had the strongest early postrest potentiation among all groups studied (rest time = 2-10 s), and SERCA2a myocytes were the least sensitive to variations in stimulation rhythm. To replicate the deficient Ca2+ removal observed in heart failure, we used 150 nM thapsigargin. Under these conditions, control myocytes exhibited slowed relaxation, whereas Parv myocytes retained their rapid kinetics, showing that Parv is still able to control relaxation, even when SERCA2a function is impaired.     

Parvalbumin isoforms differentially accelerate cardiac myocyte relaxation kinetics in an animal model of diastolic dysfunction

The cytosolic Ca(2+)/Mg(2+)-binding protein alpha-parvalbumin (alpha-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range, alpha-Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv's Mg(2+) affinity may lower the effective concentration of Parv ([Parv]) to speed relaxation and, thus, limit Parv-mediated depressed contraction. Naturally occurring alpha/beta-Parv isoforms show divergence in amino acid primary structure (57% homology) and cation-binding affinities, with beta-Parv having an estimated 16% greater Mg(2+) affinity and approximately 200% greater Ca(2+) affinity than alpha-Parv. We tested the hypothesis that, at the same or lower estimated [Parv], mechanical relaxation rate would be more significantly accelerated by beta-Parv than by alpha-Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Relaxation properties were significantly slowed in adult cardiac myocytes isolated from DS rats compared with controls: time from peak contraction to 50% relaxation was 57 +/- 2 vs. 49 +/- 2 (SE) ms (P < 0.05), validating this model system. DS cardiac myocytes were subsequently transduced with alpha- or beta-Parv adenoviral vectors. Upon Parv gene transfer, beta-Parv caused significantly faster relaxation than alpha-Parv (P < 0.05), even though estimated [beta-Parv] was approximately 10% of [alpha-Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.     

Differences in sarcoplasmic reticulum Ca2+ leak characteristics between systolic and diastolic heart failure rabbit models

Diastolic heart failure (DHF) and systolic heart failure (SHF) are two clinical subsets of chronic heart failure (CHF). Sarcoplasmic reticulum (SR) Ca2+ leak has been measured in SHF and might contribute to contractile dysfunction and arrhythmogenesis. However, no study has investigated a similar phenomenon in DHF. Thus, we established DHF and SHF rabbit models and compared the differences in Ca2+ leak between these models. New Zealand white rabbits were randomly divided into three groups (n = 8 in each group): sham operation (SO) group, DHF group and SHF group. Cardiac functions were determined by echocardiography and hemodynamic assays. The SR Ca2+ leak was measured with a calcium-imaging device and the expression and activities of related proteins were evaluated with Western blots and autophosphorylation. In the DHF group, there was significantly increased ventricular wall thickness and stiffness, reduced diastolic function, and total amount of FK506 binding protein 12.6 (FKBP12.6), increased expression and activity of protein kinase A (PKA) and phosphorylation site (P2809) in the ryanodine receptor (RyR2), but no prominent Ca2+ leak. In the SHF group, there was significantly increased ventricular cavity size, reduced systolic function, increased SR Ca2+ leak, reduced total amount of FKBP12.6 and FKBP12.6-RyR2 association, increased expression and activity of PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and their RyR2 phosphorylation sites with unchanged P2030. Our results suggest that a prominent SR Ca2+ leak was not observed in the DHF model, which may provide a new idea for the reasons in preserved systolic function, and CaMKII possibly plays a more important role in SR Ca2+ leak.     

Glycogen synthase kinase-3beta regulates growth, calcium homeostasis, and diastolic function in the heart

Glycogen synthase kinase (GSK) 3beta is a negative regulator of stress-induced cardiomyocyte hypertrophy. It is not clear, however, if GSK-3beta plays any role in regulating normal cardiac growth and cardiac function. Herein we report that a transgenic mouse expressing wild type GSK-3beta in the heart has a dramatic impairment of normal post-natal cardiomyocyte growth as well as markedly abnormal cardiac contractile function. The most striking phenotype, however, is grossly impaired diastolic relaxation, which leads to increased filling pressures of the left ventricle and massive atrial enlargement. This is due to profoundly abnormal calcium handling, leading to an inability to normalize cytosolic [Ca2+] in diastole. The alterations in calcium handling are due at least in part to direct down-regulation of the sarcoplasmic reticulum calcium ATPase (SERCA2a) by GSK-3beta, acting at the level of the SERCA2 promoter. These studies identify GSK-3beta as a regulator of normal growth of the heart and are the first of which we are aware, to demonstrate regulation of expression of SERCA2a, a critical determinant of diastolic function, by a cytosolic signaling pathway, the activity of which is dynamically modulated. De-regulation of GSK-3beta leads to severe systolic and diastolic dysfunction and progressive heart failure. Because down-regulation of SERCA2a plays a central role in the diastolic and systolic dysfunction of patients with heart failure, these findings have potential implications for the therapy of this disorder.     

Elevated cardiac tissue level of aldosterone and mineralocorticoid receptor in diastolic heart failure: Beneficial effects of mineralocorticoid receptor blocker

Cardiac aldosterone levels have not been evaluated in diastolic heart failure (DHF), and its roles in this type of heart failure remain unclear. This study aimed to detect cardiac aldosterone by use of a liquid chromatographic-mass spectrometric method and to assess the effects of mineralocorticoid receptor blockade on hypertensive DHF. Dahl salt-sensitive rats fed 8% NaCl diet from 7 wk (hypertensive DHF model) were divided at 13 wk into three groups: those treated with subdepressor doses of eplerenone (12.5 or 40 mg x kg(-1) x day(-1)) and an untreated group. Dahl salt-sensitive rats fed 0.3% NaCl diet served as controls. Cardiac aldosterone was detected in the DHF rats but not in the control rats, with increased ventricular levels of mineralocorticoid receptor. Cardiac levels of 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone were not different between the control and DHF rats, but the tissue level of corticosterone that has an affinity to mineralocorticoid receptor was 1,000 times as high as that of aldosterone. Aldosterone synthase activity and CYP11B2 mRNA were undetectable in the ventricular tissue of the DHF rats. Administration of eplerenone attenuated ventricular hypertrophy, ventricular fibrosis, myocardial stiffening, and relaxation abnormality, leading to the prevention of overt DHF. In summary, the myocardial aldosterone level increased in the DHF rats. However, its value was extremely low compared with corticosterone, and no evidence for enhancement of intrinsic myocardial aldosterone production was found. The upregulation of mineralocorticoid receptor may play a central role in the pathogenesis of DHF, and blockade of mineralocorticoid receptor is likely an effective therapeutic regimen of DHF.     

Mechanisms of impaired calcium handling underlying subclinical diastolic dysfunction in diabetes

Isolated diastolic dysfunction is found in almost half of asymptomatic patients with well-controlled diabetes and may precede diastolic heart failure. However, mechanisms that underlie diastolic dysfunction during diabetes are not well understood. We tested the hypothesis that isolated diastolic dysfunction is associated with impaired myocardial Ca(2+) handling during type 1 diabetes. Streptozotocin-induced diabetic rats were compared with age-matched placebo-treated rats. Global left ventricular myocardial performance and systolic function were preserved in diabetic animals. Diabetes-induced diastolic dysfunction was evident on Doppler flow imaging, based on the altered patterns of mitral inflow and pulmonary venous flows. In isolated ventricular myocytes, diabetes resulted in significant prolongation of action potential duration compared with controls, with afterdepolarizations occurring in diabetic myocytes (P < 0.05). Sustained outward K(+) current and peak outward component of the inward rectifier were reduced in diabetic myocytes, while transient outward current was increased. There was no significant change in L-type Ca(2+) current; however, Ca(2+) transient amplitude was reduced and transient decay was prolonged by 38% in diabetic compared with control myocytes (P < 0.05). Sarcoplasmic reticulum Ca(2+) load (estimated by measuring the integral of caffeine-evoked Na(+)-Ca(2+) exchanger current and Ca(2+) transient amplitudes) was reduced by approximately 50% in diabetic myocytes (P < 0.05). In permeabilized myocytes, Ca(2+) spark amplitude and frequency were reduced by 34 and 20%, respectively, in diabetic compared with control myocytes (P < 0.05). Sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a protein levels were decreased during diabetes. These data suggest that in vitro impairment of Ca(2+) reuptake during myocyte relaxation contributes to in vivo diastolic dysfunction, with preserved global systolic function, during diabetes.     

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.     

ACE inhibition prevents diastolic Ca2+ overload and loss of myofilament Ca2+ sensitivity after myocardial infarction

Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16- PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function.     

Role of oxidative stress in catecholamine-induced changes in cardiac sarcolemmal Ca2+ transport

Although an excessive amount of circulating catecholamines is known to induce cardiomyopathy, the mechanisms are poorly understood. This study was undertaken to investigate the role of oxidative stress in catecholamine-induced heart dysfunction. Treatment of rats for 24 h with a high dose (40 mg/kg) of a synthetic catecholamine, isoproterenol, resulted in increased left ventricular end diastolic pressure, depressed rates of pressure development, and pressure decay as well as increased myocardial Ca2+ content. The increased malondialdehyde content, as well as increased formation of conjugated dienes and low glutathione redox ratio were also observed in hearts from animals injected with isoproterenol. Furthermore, depressed cardiac sarcolemmal (SL) ATP-dependent Ca2+ uptake, Ca2+-stimulated ATPase activity, and Na+-dependent Ca2+ accumulation were detected in experimental hearts. All these catecholamine-induced changes in the heart were attenuated by pretreatment of animals with vitamin E, a well-known antioxidant (25 mg/kg/day for 2 days). Depressed cardiac performance, increased myocardial Ca2+ content, and decreased SL ATP-dependent, and Na+-dependent Ca2+ uptake activities were also seen in the isolated rat hearts perfused with adrenochrome, a catecholamine oxidation product (10 to 25 microg/ml). Incubation of SL membrane with different concentrations of adrenochrome also decreased the ATP-dependent and Na+-dependent Ca2+ uptake activities. These findings suggest the occurrence of oxidative stress, which may depress the SL Ca2+ transport and result in the development intracellular Ca2+ overload and heart dysfunction in catecholamine-induced cardiomyopathy.     

Rat cardiac contractile dysfunction induced by Ca2+ overload: possible link to the proteolysis of alpha-fodrin

The aim of the present study was to examine the mechanisms of Ca2+ overload-induced contractile dysfunction in rat hearts independent of ischemia and acidosis. Experiments were performed on 30 excised cross-circulated rat heart preparations. After hearts were exposed to high Ca2+, there was a contractile failure associated with a parallel downward shift of the linear relation between myocardial O(2) consumption per beat and systolic pressure-volume area (index of a total mechanical energy per beat) in left ventricles from all seven hearts that underwent the protocol. This result suggested a decrease in O(2) consumption for total Ca2+ handling in excitation-contraction coupling. In the hearts that underwent the high Ca2+ protocol and had contractile failure, we found marked proteolysis of a cytoskeleton protein, alpha-fodrin, whereas other proteins were unaffected. A calpain inhibitor suppressed the contractile failure by high Ca2+, the decrease in O(2) consumption for total Ca2+ handling, and membrane alpha-fodrin degradation. We conclude that the exposure to high Ca2+ may induce contractile dysfunction possibly by suppressing total Ca2+ handling in excitation-contraction coupling and degradation of membrane alpha-fodrin via activation of calpain.     

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.     

Relationship of diastolic intraventricular pressure gradients and aerobic capacity in patients with diastolic heart failure

We sought to elucidate the relationship between diastolic intraventricular pressure gradients (IVPG) and exercise tolerance in patients with heart failure using color M-mode Doppler. Diastolic dysfunction has been implicated as a cause of low aerobic potential in patients with heart failure. We previously validated a novel method to evaluate diastolic function that involves noninvasive measurement of IVPG using color M-mode Doppler data. Thirty-one patients with heart failure and 15 normal subjects were recruited. Echocardiograms were performed before and after metabolic treadmill stress testing. Color M-mode Doppler was used to determine the diastolic propagation velocity (Vp) and IVPG off-line. Resting diastolic function indexes including myocardial relaxation velocity, Vp, and E/Vp correlated well with VO2max (r = 0.8, 0.5, and -0.5, respectively, P < 0.001 for all). There was a statistically significant increase in Vp and IVPG in both groups after exercise, but the change in IVPG was higher in normal subjects compared with patients with heart failure (2.6 +/- 0.8 vs. 1.1 +/- 0.8 mmHg, P < 0.05). Increase in IVPG correlated with peak VO2max (r = 0.8, P < 0.001) and was the strongest predictor of exercise capacity. Myocardial relaxation is an important determinant of exercise aerobic capacity. In heart failure patients, impaired myocardial relaxation is associated with reduced diastolic suction force during exercise.     

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.     

Impaired S-Nitrosylation of the Ryanodine Receptor Caused by Xanthine Oxidase Activity Contributes to Calcium Leak in Heart Failure

S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca²⁺ leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca²⁺ leak, improving Ca²⁺ handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca²⁺ leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications.     

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure

Abnormal cellular Ca²⁺ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca²⁺ transient amplitude due to decreased sarcoplasmic reticulum Ca²⁺ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca²⁺ release events which occur when sarcoplasmic reticulum Ca²⁺ content exceeds a certain threshold level. Such Ca²⁺ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca²⁺ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca²⁺ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca²⁺ transients and Ca²⁺ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca²⁺ content, decreased Ca²⁺ transient amplitude, and increased frequency of Ca²⁺ waves. These results are in line with a decreased threshold for diastolic Ca²⁺ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca²⁺ release were increased THR286 phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca²⁺ content and increased propensity for diastolic Ca²⁺ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca²⁺ handling after a myocardial infarction.     

Diastolic dysfunction and diastolic heart failure: diagnostic, prognostic and therapeutic aspects

Left ventricular (LV) diastolic dysfunction (DD) and diastolic heart failure (HF), that is symptomatic DD, are due to alterations of myocardial diastolic properties. These alterations involve relaxation and/or filling and/or distensibility. Arterial hypertension associated to LV concentric remodelling is the main determinant of DD but several other cardiac diseases, including myocardial ischemia, and extra-cardiac pathologies involving the heart are other possible causes. In the majority of the studies, isolated diastolic HF has been made equal to HF with preserved systolic function (= normal ejection fraction) but the true definition of this condition needs a quantitative estimation of LV diastolic properties. According to the position of the European Society of Cardiology and subsequent research refinements the use of Doppler echocardiography (transmitral inflow and pulmonary venous flow) and the new ultrasound tools has to be encouraged for diagnosis of DD. In relation to uncertain definitions, both prevalence and prognosis of diastolic heart failure are very variable. Despite an apparent lower death rate in comparison with LV systolic HF, long-term follow-up (more than 5 years) show similar mortality between the two kinds of HF. Recent studies performed by Doppler diastolic indexes have identified the prognostic power of both transmitral E/A ratio < 1 (pattern of abnormal relaxation) and > 1.5 (restrictive patterns). The therapy of LV DD and HF is not well established but ACE-inhibitors, angiotensin inhibitors, aldosterone antagonists and β-blockers show potential beneficial effect on diastolic properties. Several trials, completed or ongoing, have been planned to treat DD and diastolic HF.