Left Ventricular Muscle Strips Research Articles

Berberine Mediated Positive Inotropic Effects on Rat Hearts via a Ca2+-Dependent Mechanism.

Previous studies showed that berberine, an alkaloid from Coptis Chinensis Franch, might exert a positive inotropic effect on the heart. However, the underlying mechanisms were unclear. Here, we reported that berberine at 10–20 µM increased the left ventricular (LV) developed pressure and the maximal rate of the pressure rising, and it increased the maximal rate of the pressure descending at 20 µM in Langendorff-perfused isolated rat hearts. These effects diminished with the concentration of berberine increasing to 50 µM. In the concentration range of 50–300 µM, berberine increased the isometric tension of isolated left ventricular muscle (LVM) strips with or without electrical stimulations, and it (30–300 µM) also increased the intracellular Ca2+ level in the isolated LV myocytes. The removal of extracellular Ca2+ hindered the berberine-induced increases in the tension of LVM strips and the intracellular Ca2+ level of LV myocytes. These suggested that berberine might exert its positive inotropic effects via enhancing Ca2+ influx. The blockade of L-type Ca2+ channels (LTCCs) with nifedipine significantly attenuated 300 μM berberine-induced tension increase in LVM strips but not the increase in the intracellular Ca2+ level. Berberine (300 μM) further increased the LVM tension following the treatment with the LTCC opener FPL-64716 (10 μM), indicating an LTCC-independent effect of berberine. Lowering extracellular Na+ attenuated the berberine-induced increases in both the tension of LVM strips and the intracellular Ca2+ level of LV myocytes. In conclusion, berberine might exert a positive inotropic effect on the isolated rat heart by enhancing the Ca2+ influx in LV myocytes; these were extracellular Na+-dependent.

Cardiac electrical and contractile disorders promoted by anabolic steroid overdose are associated with late autonomic imbalance and impaired Ca2+ handling

AimInvestigate cardiac electrical and mechanical dysfunctions elicited by chronic anabolic steroid (AS) overdose. MethodsMale Wistar rats were treated with nandrolone decanoate (DECA) or vehicle (CTL) for 8 weeks. Electrocardiography and heart rate variability were assessed at weeks 2, 4, and 8. Cardiac reactivity to isoproterenol was investigated in isolated rat hearts. Action potential duration (APD) was measured from left ventricular (LV) muscle strips. L-type Ca2+ current (ICaL), and transient outward potassium current (Ito) were recorded by whole-cell patch-clamp in LV cardiomyocytes. Sarcoplasmic reticulum (SR) Ca2+ mobilization and Ca2+-induced contractile response sensitivity were evaluated in skinned cardiac fibers. Muscarinic type 2 receptor (M2R), β1-adrenergic receptor (β1AR), sarcoplasmic Ca2+ ATPase (SERCA-2a), type 2 ryanodine receptor (RyR2), L-type Ca2+ channel (CACNA1), Kv4.2 (KCND2), and Kv4.3 (KCND3) mRNA expression levels were measured by quantitative RT-PCR. ResultsCompared with CTL group, DECA group exhibited decreased high frequency band power density (HF) and increased low frequency power density (LF), Cardiac M2R mRNA level was decreased. QTc interval at 2nd, 4th, and 8th week as well as APD30 and APD90 were increased by DECA. Ito density was decreased, while ICaL density was increased by DECA. SR Ca2+ loading and release were decreased by DECA, while contractile sensitivity to Ca2+ was increased versus CTL group. ConclusionDECA overdose induced cardiac rhythmic and mechanical abnormalities that can be associated with autonomic imbalance, up-regulated ICaL and down-regulated Ito, abnormal SR Ca2+ mobilization, and increased contractile sensitivity to Ca2+.

Increased passive stiffness promotes diastolic dysfunction despite improved Ca2+ handling during left ventricular concentric hypertrophy.

AimsConcentric hypertrophy following pressure-overload is linked to preserved systolic function but impaired diastolic function, and is an important substrate for heart failure with preserved ejection fraction. While increased passive stiffness of the myocardium is a suggested mechanism underlying diastolic dysfunction in these hearts, the contribution of active diastolic Ca2+ cycling in cardiomyocytes remains unclear. In this study, we sought to dissect contributions of passive and active mechanisms to diastolic dysfunction in the concentrically hypertrophied heart following pressure-overload.Methods and resultsRats were subjected to aortic banding (AB), and experiments were performed 6 weeks after surgery using sham-operated rats as controls. In vivo ejection fraction and fractional shortening were normal, confirming preservation of systolic function. Left ventricular concentric hypertrophy and diastolic dysfunction following AB were indicated by thickening of the ventricular wall, reduced peak early diastolic tissue velocity, and higher E/e’ values. Slowed relaxation was also observed in left ventricular muscle strips isolated from AB hearts, during both isometric and isotonic stimulation, and accompanied by increases in passive tension, viscosity, and extracellular collagen. An altered titin phosphorylation profile was observed with hypophosphorylation of the phosphosites S4080 and S3991 sites within the N2Bus, and S12884 within the PEVK region. Increased titin-based stiffness was confirmed by salt-extraction experiments. In contrast, isolated, unloaded cardiomyocytes exhibited accelerated relaxation in AB compared to sham, and less contracture at high pacing frequencies. Parallel enhancement of diastolic Ca2+ handling was observed, with augmented NCX and SERCA2 activity and lowered resting cytosolic [Ca2+].ConclusionIn the hypertrophied heart with preserved systolic function, in vivo diastolic dysfunction develops as cardiac fibrosis and alterations in titin phosphorylation compromise left ventricular compliance, and despite compensatory changes in cardiomyocyte Ca2+ homeostasis.

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction.

Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction. Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene), with dimethyl sulfoxide (DMSO)-injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels. cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. Inhibition of the RNA binding motif-20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.

Syndecan-4 is a key determinant of collagen cross-linking and passive myocardial stiffness in the pressure-overloaded heart.

Diastolic dysfunction is central to the development of heart failure. To date, there is no effective treatment and only limited understanding of its molecular basis. Recently, we showed that the transmembrane proteoglycan syndecan-4 increases in the left ventricle after pressure overload in mice and man, and that syndecan-4 via calcineurin/nuclear factor of activated T-cells (NFAT) promotes myofibroblast differentiation and collagen production upon mechanical stress. The aim of this study was to investigate whether syndecan-4 affects collagen cross-linking and myocardial stiffening in the pressure-overloaded heart. Aortic banding (AB) caused concentric hypertrophy and increased passive tension of left ventricular muscle strips, responses that were blunted in syndecan-4(-/-) mice. Disruption of titin anchoring by salt extraction of actin and myosin filaments revealed that the effect of syndecan-4 on passive tension was due to extracellular matrix remodelling. Expression and activity of the cross-linking enzyme lysyl oxidase (LOX) increased with mechanical stress and was lower in left ventricles and cardiac fibroblasts from syndecan-4(-/-) mice, which exhibited less collagen cross-linking after AB. Expression of osteopontin (OPN), a matricellular protein able to induce LOX in cardiac fibroblasts, was up-regulated in hearts after AB, in mechanically stressed fibroblasts and in fibroblasts overexpressing syndecan-4, calcineurin, or NFAT, but down-regulated in fibroblasts lacking syndecan-4 or after NFAT inhibition. Interestingly, the extracellular domain of syndecan-4 facilitated LOX-mediated collagen cross-linking. Syndecan-4 exerts a dual role in collagen cross-linking, one involving its cytosolic domain and NFAT signalling leading to collagen, OPN, and LOX induction in cardiac fibroblasts; the other involving the extracellular domain promoting LOX-dependent cross-linking.

Different Compartmentation of Responses to Brain Natriuretic Peptide and C-Type Natriuretic Peptide in Failing Rat Ventricle

We previously found a negative inotropic (NIR) and positive lusitropic response (LR) to C-type natriuretic peptide (CNP) in the failing heart ventricle. In this study, we investigated and compared the functional responses to the natriuretic peptides (NPs), brain (BNP) and C-type natriuretic peptide (CNP), and relate them to cGMP regulation and effects on downstream targets. Experiments were conducted in left ventricular muscle strips and ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. As opposed to CNP, BNP did not cause an NIR or LR, despite increasing cGMP levels. The BNP-induced cGMP elevation was mainly and markedly regulated by phosphodiesterase (PDE) 2 and was only marginally increased by PDE3 or PDE5 inhibition. Combined PDE2, -3, and -5 inhibition failed to reveal any functional responses to BNP, despite an extensive cGMP elevation. BNP decreased, whereas CNP increased, the amplitude of the Ca(2+) transient. BNP did not increase phospholamban (PLB) or troponin I (TnI) phosphorylation, Ca(2+) extrusion rate constant, or sarcoplasmatic reticulum Ca(2+) load, whereas CNP did. Both BNP and CNP reduced the peak of the L-type Ca(2+) current. Cyclic GMP elevations by BNP and CNP in cardiomyocytes were additive, and the presence of BNP did not alter the NIR to CNP or the CNP-induced PLB and TnI phosphorylation. However, a small increase in the LR to maximal CNP was observed in the presence of BNP. In conclusion, different responses to cGMP generated by BNP and CNP suggest different compartmentation of the cGMP signal and different roles of the two NPs in the failing heart.

Differential regulation of C-type natriuretic peptide-induced cGMP and functional responses by PDE2 and PDE3 in failing myocardium

Recently, we showed C-type natriuretic peptide (CNP)-induced negative inotropic (NIR) and positive lusitropic response (LR) in failing rat heart. We wanted to study whether, and if so, how phosphodiesterases (PDEs) regulate CNP-induced cyclic 3',5'-guanosine monophosphate (cGMP) elevation and functional responses. Inotropic and lusitropic responses were measured in left ventricular muscle strips and cyclic nucleotide levels, PDE activity and phospholamban (PLB) and troponin I (TnI) phosphorylation were measured in ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. CNP-mediated increase in global cGMP was mainly regulated by PDE2, as reflected by a marked amplification of the cGMP increase during PDE2 inhibition and by a high PDE2 activity in cardiomyocytes. PDE3 inhibition, on the other hand, caused no significant cGMP increase by CNP. The functional consequences did not correspond to the changes of cGMP. PDE3 inhibition increased the potency of the CNP-induced NIR and LR, while PDE2 inhibition desensitized the CNP-induced NIR, but not LR. A role for PDE2 on the maximal LR and PDE5 on the maximal NIR to CNP was revealed in the presence of PDE3 inhibition. CNP increased PLB phosphorylation about 25- to 30-fold and tended to increase TnI phosphorylation about twofold. As a whole, CNP-induced functional responses were only modestly regulated by PDEs compared to the cAMP-mediated functional responses to β1-adrenoceptor stimulation, which are highly regulated by PDEs. There is a mismatch between the CNP-induced cGMP increase and functional responses. Global cGMP levels are mainly regulated by PDE2 after CNP stimulation, whereas the functional responses are modestly regulated by both PDE2 and PDE3, indicating cGMP compartmentation by PDEs affecting CNP-induced responses in failing hearts.

Mild Hypothermia Attenuates Circulatory and Pulmonary Dysfunction During Experimental Endotoxemia*

We tested whether mild hypothermia impacts on circulatory and respiratory dysfunction during experimental endotoxemia. Randomized controlled prospective experimental study. Large animal facility, Medical University of Graz, Austria. Thirteen anesthetized and mechanically ventilated pigs. Lipopolysaccharide was administered for 4 hours. With the beginning of lipopolysaccharide infusion, animals were assigned to either normothermia (38°C, n = 7) or mild hypothermia (33°C, n = 6, intravascular cooling) and followed for 8 hours in total. At the end of the protocol, cardiac output was lower in mild hypothermia than in normothermia (4.5 ± 0.4 L/min vs 6.6 ± 0.4 L/min, p < 0.05), but systemic vascular resistance (885 ± 77 dyn·s/cm vs 531 ± 29 dyn·s/cm, p < 0.05) and (Equation is included in full-text article.)(77% ± 6% vs 54% ± 3%, p < 0.05) were higher. Indices of left ventricular contractility in vivo were not different between groups. The high-frequency band in spectral analysis of heart rate variability indicated a better preserved vagal autonomic modulation of sinuatrial node activity in mild hypothermia versus normothermia (87 ± 5 vs 47 ± 5, normalized units, p < 0.05). Plasma norepinephrine levels were elevated compared with baseline in normothermia (2.13 ± 0.27 log pg/mL vs 0.27 ± 0.17 log pg/mL, p < 0.05) but not in mild hypothermia (1.02 ± 0.31 vs 0.55 ± 0.26, p = not significant). At 38°C in vitro, left ventricular muscle strips isolated from the mild hypothermia group had a higher force response to isoproterenol. SaO2 (100% ± 0% vs 92% ± 3%, p < 0.05) and the oxygenation index (PO2/FIO2, 386 ± 52 mm Hg vs 132 ± 32 mm Hg, p < 0.05) were substantially higher in mild hypothermia versus normothermia. Plasma cytokine levels were not consistently different between groups (interleukin 10) or higher (tumor necrosis factor-α and interleukin 6 and 8) during mild hypothermia versus normothermia. The induction of mild hypothermia attenuates cardiac and respiratory dysfunction and counteracts sympathetic activation during experimental endotoxemia. This was not associated with lower plasma cytokine levels, indicating a reduction of cytokine responsiveness by mild hypothermia.

SERCA2 activity is involved in the CNP‐mediated functional responses in failing rat myocardium

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses. Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene. We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.

N‐terminal region of cardiac myosin binding protein‐C impairs myofilament function

Cardiac myosin binding protein‐C (cMyBP‐C) is a thick filament protein that plays a critical role in the regulation of sarcomere structure and contractility. Previously, we demonstrated that the N‐terminal fragment of cMyBP‐C (C0‐C1f), released during ischemia‐reperfusion (I‐R) injury, is cytotoxic and significantly impairs contractile function. However, the mechanisms in which C0‐C1f impairs cardiomyocyte contractile function remain unknown. The hypothesis of this study is that C0‐C1f depresses cardiac myofilament function. The molecular mass of C0‐C1f was analyzed by top‐down mass spectrometry. The observed molecular weights were 30974.72 Da and 31989.20 Da for mouse and human species respectively. Permeabilized left ventricular (LV) muscle strips were incubated with purified C0‐C1f. Myofilament function was characterized by measuring active tension‐[Ca2+] relationships to determine changes in maximum tension development (tmax) and Ca2+ sensitivity. Tension development and ATP consumption were simultaneously measured to determine tension‐cost over a range of [Ca2+]. C0‐C1f significantly reduced myofilament Ca2+ sensitivity and tmax, increased tension‐cost, and increased the rate of tension redevelopment (ktr). These data contribute to the understanding of contractile dysfunction following I‐R injury and provide a novel therapeutic target for the treatment of heart failure.

Increased Tension Cost in Human Familial Hypertrophic Cardiomyopathy Caused by the MYH7 Mutation R403Q

Familial hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Energy depletion of the heart is thought to initiate and promote HCM disease development. It has been hypothesized that HCM sarcomere gene mutations increase ATP utilization for myofilament contraction and thereby increase energy demand of the heart. Previous studies in single left ventricular myofibrils from myocardium harbouring the first identified causal HCM mutation R403Q in the gene encoding β-myosin heavy chain revealed increased rates of tension generation and relaxation in R403Q compared to myofibrils from healthy control myocardium. The altered kinetics observed in the mutant sample predicted a 3-fold increase in the energy cost of tension generation in R403Q sarcomeres. In the present study we investigated if tension cost was indeed increased in human myocardium harbouring the R403Q mutation.Maximal force generating capacity (Fmax) and ATP consumption (ATPmax) were simultaneously measured in Triton-permeabilized left ventricular muscle strips (n=8) from a patient carrying the R403Q mutation. Left ventricular muscle strips (n=17) from 5 HCM sarcomere mutation negative patients (HCMsmn) and strips (n=6) from 2 patients with secondary hypertrophy due to aortic stenosis (LVHao) served as a control groups. Economy of myofilament contraction is expressed as tension cost (TC), i.e. amount of ATP used during force development (ATPmax/Fmax). Both Fmax and ATPmax were significantly lower in R403Q compared to HCMsmn and LVHao. TC was significantly increased in R403Q (3.8±0.5 μmolL−1s−1/kNm−2) compared to HCMsmn and LVHao (1.6±0.1 and 1.7±0.1 μmolL−1s-1/kNm−2, respectively).Our data provide direct evidence that TC is increased in myocardium harbouring the MYH7 mutation R403Q, indicating that expression of R403Q in the heart impairs economy of myocardial contraction.Funding: Seventh Framework Program of the European Union “BIG-HEART,” grant agreement 241577

The HCM-Associated Cardiac Troponin T Mutation K280N Increases the Energetic Cost of Tension Generation in Human Cardiac Myofibrils

A novel homozygous mutation in the TNNT2 gene encoding cardiac troponin T (cTnT K280N) was identified in one HCM patient undergoing cardiac transplantation. mRNA and Mass Spectrometry analyses revealed expression of the mutant alleles without evidence of haploinsufficiency. Kinetics of contraction and relaxation of myofibrils from a frozen left ventricular sample of the K280N HCM patient were compared to those of “control” myofibrils (from donor hearts, from aortic stenosis patients, and from HCM patients negative for sarcomeric protein mutations). Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (<10 ms) solution switching. The rate constant of active tension generation following maximal Ca2+ activation (kACT) was markedly faster in K280N myofibrils (ca. 1.7 s−1) compared to controls (0.7-1 s−1). The rate constant of isometric relaxation (slow kREL) was 2-3-fold faster in K280N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the mutant preparations. The results suggest that the energy cost of tension generation is increased in the K280N sarcomeres. Simultaneous measurements of maximal isometric ATPase and Ca2+-activated force in Triton-permeabilized left ventricular muscle strips from the K280N sample demonstrated that tension cost was much higher in the K280N than in control myocardium. Replacement of the mutant protein by exchange with wild-type recombinant human Tn in the K280N myofibrils reduced both kACT and slow kREL close to control values. This indicates that the HCM-associated TNNT2 mutation K280N primarily alters apparent cross-bridge kinetics and impairs sarcomere energetics. Supported by the 7th Framework Program of the European Union, “BIG-HEART” grant agreement 241577.

Phosphatidylinositol 3,5-Bisphosphate (PI(3,5)P2) Potentiates Cardiac Contractility via Activation of the Ryanodine Receptor

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.

Phosphatidylinositol 3,5 bisphosphate increases contractility and intracellular calcium in cardiac muscle by directly activating the cardiac ryanodine receptor.

We have previously reported that treatment of left ventricular cardiac muscle strips with phosphatidylinositol 3,5 bisphosphate (PI(3,5)P2) during field stimulation enhanced the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. In addition, PI(3,5)P2 caused an increase in intracellular calcium (Ca) in cardiac myocytes. These data suggested that PI(3,5)P2 increased contractile force by promoting extracellular Ca entry or by facilitating intracellular Ca release. To elucidate the mechanism responsible for the effects of PI(3,5) on intracellular Ca, we have performed ratiometric Ca imaging in isolated adult mouse cardiac myocytes. We have found that PI(3,5)P2 induced a dramatic increase in intracellular Ca levels, which was inhibited by preincubation with ryanodine. To confirm that PI(3,5)P2 was acting on RyR2 we performed electrophysiological and biochemical studies. PI(3,5)P2 increased [3H]ryanodine binding and this effect was prevented by preincubation with an antibody against PI(3,5)P2. Furthermore, single RyR2 channels reconstituted in lipid bilayers were activated by PI(3,5)P2 in a dose‐dependent manner, indicating that PI(3,5)P2 binds directly to RyR2. We provide inaugural evidence that PI(3,5)P2 regulates cardiac contractility via modulation of intracellular Ca by direct binding and activation of the RyR2 channel.

Myofilament Force Development is Less Economic in Post-Infarct Remodeled Myocardium

Mismatch between energy supply and demand may limit cardiac performance of the remodeled heart after myocardial infarction. To assess whether economy of myofilament contraction is altered in the post-infarct remodeled myocardium, force and ATP utilization were measured simultaneously in permeabilized muscle strips from pigs with a myocardial infarction (MI). Remote left ventricular subendocardial muscle strips were taken 3 weeks after sham surgery (n=5) or induction of MI (n=5), by ligation of the left circumflex coronary artery.

Natriuretic peptides increase β1-adrenoceptor signalling in failing hearts through phosphodiesterase 3 inhibition

Whereas natriuretic peptides increase cGMP levels with beneficial cardiovascular effects through protein kinase G, we found an unexpected cardio-excitatory effect of C-type natriuretic peptide (CNP) through natriuretic peptide receptor B (NPR-B) stimulation in failing cardiac muscle and explored the mechanism. Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban (PLB) and troponin I was measured by western blotting. Stimulation of NPR-B enhanced beta1-adrenoceptor (beta1-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced beta1-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased beta1-AR-stimulated protein kinase A activity, as demonstrated by increased PLB and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts. An NPR-B-cGMP-PDE3 inhibitory pathway enhances beta(1)-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.

The effects of phosphatidylinositol 3,5‐bisphosphate on cardiac muscle

The downstream product of phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2), inositol 1,4,5‐trisphosphate (IP3), is known to induce calcium (Ca) release from the sarcoplasmic reticulum and elicit alterations in contraction of the heart and arrhythmias. What has not been studied is the effects of other phosphoinositides (PIs) such as phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2) and phosphatidylinositol 3‐phosphate (PI(3)P) on the heart. A striated muscle specific phosphatase (MIP/MTMR14) dephosphorylates PI(3,5)P2, and is present in skeletal and cardiac muscle. MIP knockout animals display reduced exercise performance, providing a strong rationale that PI(3,5)P2 may be involved in heart function. We tested 0.5 µM PI(3,5)P2, PI(4,5)P2, and PI(3)P on left ventricular muscle strips from adult mice paced at 1 Hz. All PIs tested induced an increase in the magnitude of the twitch‐force of the left ventricle. PI(3,5)P2 was also applied to single isolated cardiac myocytes and induced an increase in intracellular Ca levels and altered spontaneous Ca waves. While the mechanism of these actions remains to be determined, these data suggest that PI(3,5)P2, PI(4,5)P2, and PI(3)P may play a role in altering cardiac function. It will be essential to determine the potential role of this novel signaling mechanism in heart‐related diseases where Ca homeostasis is known to be modified.

Cardiac remodeling and dysfunction in nephrotic syndrome

There is an increased incidence of heart disease in patients with chronic nephrotic syndrome (NS), which may be attributable to the malnutrition and activated inflammatory state accompanying the sustained proteinuria. In this study, we evaluated renal function, cardiac morphometry, contractile function, and myocardial gene expression in the established puromycin aminonucleoside nephrosis rat model of NS. Two weeks after aminonucleoside injection, there was massive proteinuria, decreased creatinine clearance, and a negative sodium balance. Skeletal and cardiac muscle atrophy was present and was accompanied by impaired left ventricular (LV) hemodynamic function along with decreased contractile properties of isolated LV muscle strips. The expression of selected cytokines and proteins involved in calcium handling in myocardial tissue was evaluated by real time polymerase chain reaction. This revealed that the expression of interleukin-1beta, tumor necrosis factor-alpha, and phospholamban were elevated, whereas that of cardiac sarco(endo)plasmic reticulum calcium pump protein was decreased. We suggest that protein wasting and systemic inflammatory activation during NS contribute to cardiac remodeling and dysfunction.

Myocardial dysfunction and neurohumoral activation without remodeling in left ventricle of monocrotaline-induced pulmonary hypertensive rats

In monocrotaline (MCT)-induced pulmonary hypertension (PH), only the right ventricle (RV) endures overload, but both ventricles are exposed to enhanced neuroendocrine stimulation. To assess whether in long-standing PH the left ventricular (LV) myocardium molecular/contractile phenotype can be disturbed, we evaluated myocardial function, histology, and gene expression of autocrine/paracrine systems in rats with severe PH 6 wk after subcutaneous injection of 60 mg/kg MCT. The overloaded RV underwent myocardial hypertrophy (P < 0.001) and fibrosis (P = 0.014) as well as increased expression of angiotensin-converting enzyme (ACE) (8-fold; P < 0.001), endothelin-1 (ET-1) (6-fold; P < 0.001), and type B natriuretic peptide (BNP) (15-fold; P < 0.001). Despite the similar upregulation of ET-1 (8-fold; P < 0.001) and overexpression of ACE (4-fold; P < 0.001) without BNP elevation, the nonoverloaded LV myocardium was neither hypertrophic nor fibrotic. LV indexes of contractility (P < 0.001) and relaxation (P = 0.03) were abnormal, however, and LV muscle strips from MCT-treated compared with sham rats presented negative (P = 0.003) force-frequency relationships (FFR). Despite higher ET-1 production, BQ-123 (ET(A) antagonist) did not alter LV MCT-treated muscle strip contractility distinctly (P = 0.005) from the negative inotropic effect exerted on shams. Chronic daily therapy with 250 mg/kg bosentan (dual endothelin receptor antagonist) after MCT injection not only attenuated RV hypertrophy and local neuroendocrine activation but also completely reverted FFR of LV muscle strips to positive values. In conclusion, the LV myocardium is altered in advanced MCT-induced PH, undergoing neuroendocrine activation and contractile dysfunction in the absence of hypertrophy or fibrosis. Neuroendocrine mediators, particularly ET-1, may participate in this functional deterioration.

Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy.

The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly (P<0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (n=7), intermediate in those with pseudonormal filling (n=6), and lowest in the group with restrictive filling (n=7). Mechanical measurements on LV muscle strips dissected from these hearts (n=8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O2 consumption (n=10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.