Cardiomyocyte Relaxation Research Articles

Life-Long Treatment with Late Sodium Current Blocker Reduces Myocardial Dysfunction and Remodeling in a Mouse Model of Hypertrophic Cardiomyopathy

No drugs are capable to prevent phenotype development and adverse cardiac remodeling in hypertrophic cardiomyopathy (HCM). Ranolazine, a late Na+ current blocker, reduced arrhythmogenicity and improved relaxation in cardiomyocytes and trabeculae from HCM patients (Coppini et al., Circulation 2013). Employing a transgenic mouse model carrying the HCM-associated R92Q mutation in the TNNT2 gene, we previously showed that acute in vitro treatment with ranolazine is capable to reverse some electromechanical alterations, including the prolonged kinetics of Ca2+ transients, the higher diastolic [Ca2+] and the increased frequency of arrhythmogenic spontaneous activity (Pioner et al., Biophys J 2014, 106, 644a). Here we employed the same mouse model to assess whether long-term oral treatment with ranolazine since birth is capable to prevent the HCM phenotype and the associated myocardial remodeling. We compared the behavior of WT, R92Q-untreated and R92Q-treated 1 year old mice. Echocardiographic measurements showed that the R92Q-treated in vivo hearts lacked the left ventricular hypertrophy, hypercontractility and diastolic dysfunction found in the R92Q-untreated mice. Gadolinium-contrast magnetic resonance showed that the intramyocardial fibrosis of the R92Q-untreated hearts was largely reduced in the treated mice.Both amelioration of cardiomyocyte function and reduction of extracellular fibrosis may contribute to the positive effect of the long-term treatment with ranolazine that could represent a candidate for preventive treatment of phenotype-negative mutation carriers. Mechanical experiments in intact left and right ventricular trabeculae confirmed the alterations we had previously reported in R92Q-untreated mice compared to WT and showed that those alterations were mostly reversed in the R92Q-treated mice. In the R92Q-treated preparations the inotropic response to isoproterenol was preserved and the occurrence of spontaneous activity was markedly reduced compared to the untreated trabeculae and was comparable to that of WT trabeculae.

Abstract 13407: A Mouse Model of Heart Failure with Preserved Ejection Fraction (HFpEF) due to Chronic Infusion of a Low Subpressor Dose of Angiotensin II

Introduction: Heart failure with preserved ejection fracture (HFpEF) is a clinical syndrome of HF symptoms associated with impaired diastolic function. Although it represents approximately 50% of all patients with HF, the mechanisms of disease are poorly understood, animal models of HFpEF not due pressure overload are lacking, and therapies for HFpEF are generally ineffective. Hypothesis: A continuous infusion of low dose of angiotensin II (ATII) may be sufficient to induce changes in left ventricular (LV) diastolic function without increasing blood pressure nor induce LV hypertrophy. Methods: Osmotic pumps were implanted subcutaneously in 8 week-old CD1 male mice randomly assigned to the ATII (200 ng/kg/day) or vehicle (N=8/group). Transthoracic echocardiography was performed at baseline and 4 weeks to measure LV dimensions, systolic and diastolic function. Aortic systolic and diastolic pressures (AoP), LV peak systolic and end-diastolic pressures (LVPSP, LVEDP) were measured at LV catheterization. Fibrosis was measured using Masson’s trichrome stain. The expression of Interleukin (IL)-18 mRNA levels, a cytokine associated with impaired cardiomyocyte relaxation, was measured at 4 weeks. Results: When compared to the baseline values or vehicle group, ATII infusion had no effects on AoP, LVPSP and HR, and had no effects on LV dimensions or mass, nor on LVEF (all P>0.2). ATII induced a significant impairment in LV diastolic function as measured by an increase (worsening) of the myocardial performance index (MPI), the LV isovolumetric relaxation time (IVRT) and of LVEDP (Figure). Cardiac expression of IL-18 mRNA was significantly increased 7-fold after ATII infusion (P<0.001), suggesting a potential mechanistic role of IL-18. Conclusion: Chronic infusion of low dose ATII recapitulates the HFpEF phenotype in the mouse, without increasing blood pressure. The use of this mouse model may help understanding the mechanisms leading to HFpEF syndrome in patients.

Dynamic Changes in Myocardial Matrix and Relevance to Disease

The cardiac extracellular matrix (ECM) provides the architectural scaffold to support efficient contraction and relaxation of cardiomyocytes. The elegant design of the ECM facilitates optimal force transduction, electric transmission, intercellular communication, and metabolic exchange within the myocardial microenvironment. In the setting of increased wall stress, injury, or disease, the ECM can undergo a series of dynamic changes that lead to favorable chamber remodeling and functional adaptation. Over time, sustained matrix remodeling can impair diastolic and systolic function caused by excess deposition of interstitial fibrous tissue. These pathological alterations in ECM structure/function are considered central to the evolution of adverse cardiac remodeling and the development of heart failure. This review discusses the complex dynamics of the cardiac ECM in the setting of myocardial infarction, pressure overload, and volume overload. We also summarize the current status of ECM biomarkers that may have clinical value in prognosticating cardiac disease progression in patients. Finally, we discuss the most current status of drugs under evaluation for use in cardiac fibrosis.

CrossTalk opposing view: The late sodium current is not an important player in the development of diastolic heart failure (heart failure with a preserved ejection fraction)

CrossTalk opposing view: The late sodium current is not an important player in the development of diastolic heart failure (heart failure with a preserved ejection fraction)

Ranolazine improves diastolic function in spontaneously hypertensive rats

Diastolic dysfunction can lead to heart failure with preserved ejection fraction, for which there is no effective therapeutic. Ranolazine has been reported to reduce diastolic dysfunction, but the specific mechanisms of action are unclear. The effect of ranolazine on diastolic function was examined in spontaneously hypertensive rats (SHRs), where left ventricular relaxation is impaired and stiffness increased. The objective of this study was to determine whether ranolazine improves diastolic function in SHRs and identify the mechanism(s) by which improvement is achieved. Specifically, to test the hypothesis that ranolazine, by inhibiting late sodium current, reduces Ca(2+) overload and promotes ventricular relaxation and reduction in diastolic stiffness, the effects of ranolazine or vehicle on heart function and the response to dobutamine challenge were evaluated in aged male SHRs and Wistar-Kyoto rats by echocardiography and pressure-volume loop analysis. The effects of ranolazine and the more specific sodium channel inhibitor tetrodotoxin were determined on the late sodium current, sarcomere length, and intracellular calcium in isolated cardiomyocytes. Ranolazine reduced the end-diastolic pressure-volume relationship slope and improved diastolic function during dobutamine challenge in the SHR. Ranolazine and tetrodotoxin also enhanced cardiomyocyte relaxation and reduced myoplasmic free Ca(2+) during diastole at high-stimulus rates in the SHR. The density of the late sodium current was elevated in SHRs. In conclusion, ranolazine was effective in reducing diastolic dysfunction in the SHR. Its mechanism of action, at least in part, is consistent with inhibition of the increased late sodium current in the SHR leading to reduced Ca(2+) overload.

Inhibition of the NA+/CA2+ Exchanger (Ncx) Improves Cardiomyocyte Contractile Dysfunction in a Rat Model with Compensated Renal Failure and Heart Failure with Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFPEF) and renal impairment are often associated but the underlying pathomechanisms are largely unknown. We investigated tissue fibrosis and cardiomyocyte contractile function in a rat model with HFPEF and compensated renal failure.Methods: Seventy young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). Blood/urine samples, echocardiography, pressure-volume loops were performed after 8 and 24 weeks. Left ventricular (LV) hypertrophy, fibrosis (picosirius-red-staining) and NCX protein expression (Western blot) were determined after sacrification. Cardiomyocyte function (Ca2+ transients, cell shortening) was quantified in isolated cardiomyocytes without and with the NCX inhibitor SEA0400 (300nM).Results: NXT rats showed stable renal impairment with preserved urine excretion and elevated arterial pressure (148±8 and 154±7 vs. 105±3 and 109±2 mmHg, p<0.01) at 8 and 24 weeks, respectively. LV in NXT were significantly hypertrophied at 8 and 24 weeks, but LV systolic function (EF, dP/dt) were preserved. LVEDP, LA size and lung weight were significantly increased indicating HFPEF with pulmonary congestion. Fibrosis was increased in LV from NXT. LV cardiomyocytes showed significantly prolonged time for early (50%) relaxation and decay of the Ca2+ transient. Time constant of the caffeine-induced Ca2+ transient (TAU) was significantly prolonged indicating reduced NCX forward mode activity, while NCX protein expression was upregulated. Acute treatment with SEA0400 (300nM) significantly accelerated early relaxation in cardiomyocytes from NXT (8 weeks).Conclusion: In this model of compensated renal failure and HFPEF cardiomyocyte relaxation was prolonged already at an early stage. Our results suggest increased NCX activity may contribute to contractile dysfunction, likely through reverse mode Ca2+ influx. NCX represents a potential target to treat cardiomyocyte dysfunction in HFPEF.

Cardiovascular and metabolic effects of vitamin D

In humans, approximately 95% of the total requirement for vitamin D is synthesized in the skin by the effects of sunlight. The active vitamin D synthesis from 25 (OH) D takes place via the 1-alpha hydroxylase enzyme both in renal and non-renal tissues. The effect of vitamin D on the receptor level is through active vitamin D. Vitamin D receptors are shown in more than 30 tissues. Non-classical effects of vitamin D are grouped under three main headings: Regulation of hormone secretion, regulation of immune function, regulation of cell proliferation and differentiation. Cardiovascular diseases, renal diseases, diabetes, obesity, metabolic syndrome are mostly researched diseases related with vitamin D. Vitamin D levels were shown inversely to be related with hypertension, obesity, diabetes and hypertriglyceridemia. In vitro studies have shown that vitamin D accelerates relaxation in cardiomyocytes and improves diastolic function of the heart. In clinical studies done in patients with chronic kidney disease, poor vitamin D status was shown to be an independent risk factor for coronary vascular calcification, other heart diseases such as heart failure, and cardiovascular mortality. In this review, the cardiovascular and metabolic effects of vitamin D have been reviewed in the light of current knowledge.

2-deoxy-ATP Alters Myosin Structure to Enhance Cross-Bridge Cycling and Improve Cardiac Function

We previously reported that increased cellular 2-deoxy-ATP (dATP) augments contraction, enhancing the rate and magnitude of both contraction and relaxation in intact cardiomyocytes, force production in skinned cardiac muscle, and fractional shortening of the whole heart. To better understand the mechanism by which dATP enhances cardiac function, we used molecular dynamics (MD) simulations to study the pre- and post-powerstroke states of myosin (PDB ID: 1VOM and 1MMA) bound to either Mg2+.(d)ADP.Pi (pre-powerstroke) or Mg2+.(d)ADP (post-powerstroke) for 50ns. In both states, dATP binding to myosin alters the conformation of the nucleotide binding pocket and the actin binding surface. In the pre-powerstroke state, myosin made fewer contacts with dADP than with ADP and this structural change translated to increased exposure of polar residues on the actin-binding surface of myosin. Since the initial acto-myosin interaction is primarily electrostatic, the affinity of myosin for actin may thus be enhanced with dADP.Pi. In post-powerstroke simulations, dADP binding reduced the exposure of actin binding residues on myosin somewhat. This may translate into improved ability for myosin detachment from actin at the end of the powerstroke in the presence of dADP vs. ADP. Furthermore, stopped-flow spectroscopy demonstrated that myosin has 40-80% weaker binding affinity for dADP than ADP, which may also contribute to faster myosin detachment. Together, these data demonstrate that dADP binding to myosin significantly alters the conformation of myosin, which may translate to faster cross-bridge cycling due to both enhanced myosin binding in the pre-powerstroke state and faster myosin detachment from actin in the post-powerstroke state. Ongoing studies examining the effect of enhanced cross-bridge cycling with elevated dATP in infarcted hearts suggest that dATP may reduce the loss of systolic function. Supported by HL111197 (MR), WT085309 (MAG), GM50789 (VD) and T32EB001650 (SGN).

Compensated Cardiomyocyte Ca2+ Homeostasis in a Rat Model of Diastolic Heart Failure

In approximately half of heart failure patients, systolic function is near normal while diastolic function is impaired. This condition, called diastolic heart failure (DHF), or heart failure with preserved ejection fraction (HFPEF), has been reported to result from reduced passive distensibility of the ventricle, which impairs filling. However, the contribution of altered Ca2+ handling to impaired cardiomyocyte relaxation in this condition remains largely uninvestigated. We examined this issue in a rat model of DHF following aortic stenosis. 6 weeks after aortic banding, cardiac function was characterized by echocardiography, and rats exhibiting systolic heart failure (left atrial diameter >5mm, left ventricular dilation and reduced fractional shortening) were excluded. Echo Doppler measurements revealed decreased peak early diastolic tissue velocity in DHF rats compared to sham-operated controls (64±5 vs 85±5 mm/s, P<0.05). Both heart and lung weights were increased in DHF animals, indicating hypertrophy and congestion. In isolated cardiomyocytes, Ca2+ transients recorded by whole-cell fluorescence (fluo 4-AM) during field stimulation were of larger magnitude in DHF than control cells, across a range of frequencies (0.5-6 Hz). Ca2+ transients also decayed more rapidly in DHF, and at high frequencies exhibited elevated SR content and a smaller increase in diastolic [Ca2+]. Such augmentation of Ca2+ homeostasis was consistent with an increase in both sarcoplasmic reticulum Ca2+ reuptake and Ca2+ extrusion in DHF cells. Confocal imaging of cells stained with di-8-ANEPPs indicated maintained t-tubule organization, consistent with an unaltered time to peak of the Ca2+ transients. Thus, contrary to impaired Ca2+ homeostasis widely reported in systolic heart failure, our results suggest that DHF develops despite improved Ca2+ handling. We postulate that other alterations such as increased passive ventricular stiffness or increased myofilament Ca2+ sensitivity must impair diastolic function in this condition.

Molecular Details of SERCA Regulation by Phospholamban Revealed by Paramagnetic Relaxation Enhancements and Solid-State NMR

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) ia a 110 kDa membrane protein that regulates cardiomyocyte relaxation by transporting Ca2+ into the SR. SERCA is regulated by PLN, a 52-resdiue membrane protein consisting of a helical, inhibitory transmembrane domain and a regulatory cytoplasmic domain. The cytoplasmic domain is in equilibrium between a folded T state and an unfoled R state. Phosphorylation of PLN at S16 relieves the inhibition of SERCA and, based on studies in animal models, mimicking the effects of PLN phosphorylation is a promising path for the development of therapeutics against heart failure.Here we co-reconstituted SERCA and PLN in lipid bilayers under fully functional conditions and utilized solid state NMR to probe the molecular mechanisms of SERCA regulation. Asymmetric spin labeling and isotopic labeling of SERCA, PLN and lipids in combination with magic angle spinning experiments provided paramagnetic relaxation enhancements (PREs) that mapped the structure of the membrane protein complex. PREs were combined with oriented restraints from separated local field experiments, chemical shift perturbations and torsion angles from backbone chemical shifts to determine a structural model of the PLN/SERCA complex. The transmembrane domain of PLN remains anchored to the ATPase, while the cytoplasmic domain is membrane-associated in the T state and interacts transiently with the phosphorylation and nucleotide domains of SERCA in the R state. Phosphorylation increases the R state population and the affinity of the R state for SERCA. These results explain the non-inhibitory character of PLN mutants with increased R state population and provide crucial information for future design of therapeutics to increase SERCA function. In addition, the methodological advances described here further open the landscape to conducting structural studies of membrane protein complexes of molecular weights beyond 100 kDa.

Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform

It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG) and that exposure of zinc ion (Zn2+) to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR) at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our results suggest that the relaxing effects of Zn2+ on cardiomyocyte function are more pronounced in the HG state due an insulin-dependent effect of enhancing removal of cytosolic Ca2+ via SERCA2a or NCX or by reducing Ca2+ influx via L-type channel or Ca2+ leak through the RyR. Investigations into the effects of Zn2+ on these mechanisms are now underway.

Broad Transgenic, and Cardiac-Specific Viral Mediated, Over-Expression of Ribonucleotide Reductase Increases In Vivo Cardiac Contractility

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all activating [Ca2+] in demembranated cardiac muscle via increased crossbridge binding and cycling rates. Furthermore, we demonstrated that cellular [dATP] is increased by adenoviral-driven overexpression of the ribonucleotide reductase (Rrm) complex, which increased contractility with no decrement of relaxation in adult rat cardiomyocytes from normal or infarcted hearts. In this study we hypothesized that in vivo overexpression of Rrm would increase cardiac contractility. Elevated cardiac Rrm was accomplished by 1) transgenic (Tg) overexpression in mice via the chicken beta-actin promoter and 2) intravenous delivery of recombinant adeno-associated virus (rAAV6) to drive Rrm production using a cardiac specific promoter (cTnT455). Echocardiographic fractional shortening was significantly increased in 3-month old Tg animals (43.5 ± 1.4%) as compared to wild-type (WT) littermates (34.5 ± 2.2%), and this increase persisted at 6 months of age (42.8 ± 2.5% vs 28.8 ± 2.7%). Similar increases in FS were seen in rAAV6-Rrm transduced animals (43.0 ± 2.1%) as compared to sham-injected control animals (32.1 ± 2.9%). LV pressure development (balloon catheterization) was elevated in Tg-Rrm vs. WT Langendorff perfused hearts, and concurrent NMR spectra indicated no difference in PCr:ATP ratio during high workload challenge. Upregulation of Rrm was confirmed via RT-PCR, western blot analysis, and immunohistochemistry. Preliminary western blot results also suggest no difference in SERCA expression, but increased Ser-16 phosphorylation of phospholamban, in Tg-Rrm vs WT hearts. This may, in part, explain our previously reported acceleration of Ca2+ transient decay seen in Rrm-overexpressing cardiomyocytes in vitro. Overall, these experiments demonstrate the feasibility of directly targeting the actin-myosin crossbridge to enhance cardiac contractility without impairing relaxation. HL65497, HL091368, AHA2310117.

Treinamento em natação atenua a disfução contrátil de cardiomiócitos de ratos diabéticos

Experimental diabetes promotes contractile dysfunction in cardiomyocytes, but the effects of swimming in this disorder are not known.To test the effects of a swimming training program (STP) on cardiomyocyte contractile dysfunction in rats with experimental diabetes.Wistar rats (age: 30 days; mean body weight: 84.19 g) with diabetes induced by streptozotocin (60 mg/kg body weight; glucose > 300 mg/dl) were divided into sedentary diabetic rats (SD, n = 10) and exercised diabetic rats (ED, n = 13). Animals of same age and weight served as sedentary controls (SC, n = 10) and exercised controls (EC, n = 06). Animals and ED and EC underwent a STP (05 days/week, 90 min/day) for 08 weeks. Left ventricular (LV) myocytes were isolated and electrically stimulated at 3.0 Hz at room temperature (∼ 25º C).Diabetes reduced contractile function in cardiomyocytes of animals compared to controls (i.e., lower amplitude of contraction, longer duration of contraction and relaxation). The STP attenuated the reduced amplitude of contraction (SC, 11 ± 0.2% vs ED, 11.6 ± 0.2%), time to peak contraction (SC, 319 ± 5.8 ms vs ED, 333 ± 4.8 ms) and time to 50.0% of relaxation (SC, 619 ± 22.2 ms vs ED 698 ± 18.6 ms) of cardiomyocytes of diabetic rats. Diabetes reduced the size of cardiomyocytes, however, the STP minimized the reduction of cell volume and width, without changing length.The swimming training program attenuated the contractile dysfunction of the LV myocytes of rats with experimental diabetes.

The influence of trout cardiac troponin I and PKA phosphorylation on the Ca2+ affinity of the cardiac troponin complex

The trout heart is 10-fold more sensitive to Ca(2+) than the mammalian heart. This difference is due, in part, to cardiac troponin C (cTnC) from trout having a greater Ca(2+) affinity than human cTnC. To determine what other proteins are involved, we cloned cardiac troponin I (cTnI) from the trout heart and determined how it alters the Ca(2+) affinity of a cTn complex containing all mammalian components (mammalian cTn). Ca(2+) activation of the complex was characterized using a human cTnC mutant that contains anilinonapthalenesulfote iodoacetamide attached to Cys53. When the cTn complex containing labeled human cTnC was titrated with Ca(2+), its fluorescence changed, reaching an asymptote upon saturation. Our results reveal that trout cTnI lacks the N-terminal extension found in cTnI from all other vertebrate groups. This protein domain contains two targets (Ser23 and Ser24) for protein kinase A (PKA) and protein kinase C. When these are phosphorylated, the rate of cardiomyocyte relaxation increases. When rat cTnI in the mammalian cTn complex was replaced with trout cTnI, the Ca(2+) affinity was increased ∼1.8-fold. This suggests that trout cTnI contributes to the high Ca(2+) sensitivity of the trout heart. Treatment of the two cTn complexes with PKA decreased the Ca(2+) affinity of both complexes. However, the change for the complex containing rat cTnI was 2.2-fold that of the complex containing trout cTnI. This suggests that the phosphorylation of trout cTnI does not play as significant a role in regulating cTn function in trout.

Carbon Monoxide Pollution Promotes Cardiac Remodeling and Ventricular Arrhythmia in Healthy Rats

Epidemiologic studies associate atmospheric carbon monoxide (CO) pollution with adverse cardiovascular outcomes and increased cardiac mortality risk. However, there is a lack of data regarding cellular mechanisms in healthy individuals. To investigate the chronic effects of environmentally relevant CO levels on cardiac function in a well-standardized healthy animal model. Wistar rats were exposed for 4 weeks to filtered air (CO < 1 ppm) or air enriched with CO (30 ppm with five peaks of 100 ppm per 24-h period), consistent with urban pollution. Myocardial function was assessed by echocardiography and analysis of surface ECG and in vitro by measuring the excitation-contraction coupling of single left ventricular cardiomyocytes. Chronic CO pollution promoted left ventricular interstitial and perivascular fibrosis, with no change in cardiomyocyte size, and had weak, yet significant, effects on in vivo cardiac function. However, both contraction and relaxation of single cardiomyocytes were markedly altered. Several changes occurred, including decreased Ca(2+) transient amplitude and Ca(2+) sensitivity of myofilaments and increased diastolic intracellular Ca(2+) subsequent to decreased SERCA-2a expression and impaired Ca(2+) reuptake. CO pollution increased the number of arrhythmic events. Hyperphosphorylation of Ca(2+)-handling and sarcomeric proteins, and reduced responses to beta-adrenergic challenge were obtained, suggestive of moderate CO-induced hyperadrenergic state. Chronic CO exposure promotes a pathological phenotype of cardiomyocytes in the absence of underlying cardiomyopathy. The less severe phenotype in vivo suggests a role for compensatory mechanisms. Arrhythmia propensity may derive from intracellular Ca(2+) overload.

Factors controlling the activity of the SERCA2a pump in the normal and failing heart

Heart failure is the leading cause of death in western countries and is often associated with impaired Ca(2+) handling in the cardiomyocyte. In fact, cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (ER/SR) Ca(2+) pump SERCA2a, pumping Ca(2+) from the cytosol into the lumen of the ER/SR. This review addresses three important facets that control the SERCA2 activity in the heart. First, we focus on the alternative splicing of the SERCA2 messenger, which is strictly regulated in the developing heart. This splicing controls the formation of three SERCA2 splice variants with different enzymatic properties. Second, we will discuss the role and regulation of SERCA2a activity in the normal and failing heart. The two well-studied Ca(2+) affinity modulators phospholamban and sarcolipin control the activity of SERCA2a within a narrow window. An aberrantly high or low Ca(2+) affinity is often observed in and may even trigger cardiac failure. Correcting SERCA2a activity might therefore constitute a therapeutic approach to improve the contractility of the failing heart. Finally, we address the controversies and unanswered questions of other putative regulators of the cardiac Ca(2+) pump, such as sarcalumenin, HRC, S100A1, Bcl-2, HAX-1, calreticulin, calnexin, ERp57, IRS-1, and -2.

SPOC: A Functional Assay of Failing and Non-Failing Human Cardiomyocytes

SPontaneous Oscillatory Contractions, known as SPOCs, are well characterized for skeletal muscle fibers but less so for cardiac muscle. A survey of SPOCs on mammalian hearts from rat to cow showed that, like myosin ATPase activity, SPOC contractions exhibit a linear relationship relative to body size. SPOCs exhibit slow, travelling waves of relaxations that can be induced under precise solvent conditions. These are observed by light microscopy and the time-resolved images are analysed with high spatial and temporal resolution for up to 1 h. Here we examine the functional performance of failing and non-failing human heart samples snap frozen at -200°C (liquid nitrogen) within minutes of clamping the coronary arteries. Thus, it is possible to study the contraction and relaxation of cardiomyocytes from hearts that were also used in transcriptomics studies. With our large bank of frozen human heart tissue we can study the effects of: (1) end-stage heart failure on multiple heart samples from a wide range of patients; and (2) aging on contractile performance using multiple samples of non-failing hearts. We will report preliminary findings from end-stage heart failure patients with familial and non-familial dilated cardiomyopathy, and from un-used donor hearts aged 9-65 years.

Extracellular Zinc Enhances Cardiomyocyte Relaxation Function in Diabetic Rats

Diabetes mellitus (DM) leads to a cardiomyopathy in humans and rodent models. Interestingly, the chronic infusion of zinc in DM mice prevents the development of the cardiomyopathy. To elucidate the possible mechanism underlying this observation, we examined the effects of extracellular zinc ion (Zn2+) on cardiomyocyte function in DM rats (n=5) compared to non-DM controls (Ctrl, n=5). Both the DM and Ctrl rats were hypothyroid, which assured similar upregulation of β-myosin heavy chain (β-MHC) in both populations. Isolated cardiomyocytes were electrically stimulated at 2, 4, 6 and 6.5 Hz, maintained at 35°C and exposed to 1.2 mM extracellular Ca2+. Sarcomere shortening and relengthening dynamics were monitored using a video-based Fourier-transform technique. Without extracellular Zn2+ peak shortening as a fraction of diastolic sarcomere length was statistically greater (P<0.05) in the DM (6.56 ± 0.99 %; n = 16) compared to Ctrl (5.27 ± 1.91 %; n = 20) at 2 Hz, but not at higher frequencies. Time to 50% return to diastolic sarcomere length was not statistically different between the groups at every pacing frequency. Exposure to 12 μM extracellular Zn2+ significantly reduced (P<0.001) peak shortening in both the DM and Ctrl at all frequencies. There was a strong trend (P = 0.070) toward Zn2+ exposure significantly shortening the time to 50% return in the DM but not in the Ctrl, as revealed by repeated-measures ANOVA. Similar trends were found for time to peak shortening (P=0.087) and time to 10% return (P=0.048). These results suggest that extracellular Zn2+ inhibits cardiomyocyte contractile function independent of DM, perhaps by competing with intracellular Ca2+, and enhances cardiomyocyte diastolic function in the DM. The enhanced sensitivity of the DM to the relaxing effects of Zn2+ may underlie the protective effects of Zn2+ against diabetic cardiomyopathy.

Prevention of endotoxin-induced sarcoplasmic reticulum calcium leak improves mitochondrial and myocardial dysfunction*

Growing evidence suggests that mitochondria function is impaired in sepsis. Here, we tested the hypothesis that lipopolysaccharide would induce mitochondrial Ca2+ overload and oxygen utilization abnormalities as consequences of sarcoplasmic reticulum Ca2+ handling derangements that are typically observed in sepsis. As lipopolysaccharide-induced sarcoplasmic reticulum dysfunction was mainly characterized by reduced sarcoplasmic reticulum Ca2+ uptake and Ca2+ leak, we tested whether dantrolene, a sarco(endo)plasmic reticulum calcium ATPase leak inhibitor, would prevent mitochondrial and cardiac contractile dysfunction. Randomized controlled trial. Experimental laboratory. Male Sprague Dawley rats. Sepsis was induced by injection of endotoxin lipopolysaccharide (10 mg/kg/intravenously). Assessment of contractile function and Ca2+ handling was performed 4 hr after lipopolysaccharide. The relative contribution of the different Ca2+ transporters to relaxation in intact cardiomyocytes was studied during successive electrically evoked twitches and caffeine stimulation. Sarcoplasmic reticulum vesicles and mitochondria from ventricles of rats treated or not with lipopolysaccharide were prepared to evaluate Ca2+ uptake-release and oxygen fluxes, respectively. Effects of dantrolene (10 mg/kg) treatment in rats were evaluated in sarcoplasmic reticulum vesicles, mitochondria, and isolated hearts. Lipopolysaccharide challenge elicited cardiac contractile dysfunction that was accompanied by severe derangements in sarcoplasmic reticulum function, i.e., reduced Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Functional sarcoplasmic reticulum changes were associated with modification in the status of phospholamban phosphorylation whereas SERCA was unchanged. Rises in mitochondrial Ca2+ content observed in lipopolysaccharide-treated rats coincided with derangements in mitochondrial oxygen efficacy, i.e., reduced respiratory control ratio. Administration of dantrolene in lipopolysaccharide-treated rats prevented mitochondrial Ca2+ overload and mitochondrial oxygen utilization abnormalities. Moreover, dantrolene treatment in lipopolysaccharide rats improved heart mitochondrial redox state and myocardial dysfunction. These experiments suggest that sarcoplasmic reticulum Ca2+ handling dysfunction is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction.

Regulation of Ryanodine Receptors in the Heart

See related article, pages 819–829 In response to exercise or other stresses, catecholamines are released into the circulation and within the heart. Catecholaminergic stimulation of β1-adrenergic receptors (β1ARs) in the heart increases heart rate (chronotropy) and contractility (inotropy), resulting in increased cardiac output during acute stress. Concurrent stimulation of β2-ARs dilates blood vessels, which increase blood flow to exercising muscles, thereby matching increased cardiac output to metabolic demands of the organs. The inotropic mechanisms investigated by Valdivia and colleagues1 in the current issue of Circulation Research are essential for the acute stress-dependent increase of cardiac output. Each of the millions of muscle cells in the heart (cardiomyocytes) contribute to myocardial force development. Cardiomyocyte contraction is controlled by intracellular Ca2+ release through a process called excitation-contraction coupling (ECC) that involves the following steps: (1) an action potential (AP) depolarizes the cell membrane; (2) voltage-dependent plasma membrane L-type calcium channels (Cav1.2) opening results in a whole-cell inward Ca2+ current ( I Ca); (3) I Ca activates cardiac ryanodine receptor (RyR2)/Ca2+ release channels located on the junctional sarcoplasmic reticulum (jSR), a process referred to as Ca2+-induced Ca2+ release (CICR); (4) Ca2+ binds to troponin C (TnC) leading to cross-bridge formation between myosin and actin and contraction of the sarcomere. Cardiomyocyte relaxation is signaled by a return of intracellular [Ca2+] to resting levels attributable to the following major mechanisms: (1) Cav1.2 inactivation; (2) RyR2 inactivation; (3) Ca2+ reuptake into the SR by SERCA2a pumps; and (4) Na+/Ca2+ exchange extrusion of Ca2+ to the extracellular space. Under resting conditions, net SR Ca2+ release contributes approximately 66% of Ca2+ necessary for myofilament activation in large mammals including humans, and approximately 90% in rodents.2 Stimulation …