SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.     

Calcium-calmodulin dependent protein kinase II (CaMKII): a main signal responsible for early reperfusion arrhythmias

To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca²⁺-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31 ± 6 to 5 ± 1 beats/3 min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0 ± 14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca²⁺ channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca²⁺ channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca²⁺ channels might contribute to reperfusion-induced PBs, by increasing SR Ca²⁺ content and Ca²⁺ influx.     

Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling

The histidine-rich calcium binding protein (HRC) is a novel regulator of sarcoplasmic reticulum (SR) Ca²⁺-uptake, storage and release. Residing in the SR lumen, HRC binds Ca²⁺ with high capacity but low affinity. In vitro phosphorylation of HRC affects ryanodine affinity of the ryanodine receptor (RyR), suggesting a functional role of HRC on SR Ca²⁺-release. Indeed, acute HRC overexpression in isolated rodent cardiomyocytes decreases Ca²⁺-induced Ca²⁺-release, increases SR Ca²⁺-load, and impairs contractility. The HRC effects on RyR may be regulated by the Ca²⁺-sensitivity of its interaction with triadin. However, HRC also affects the SR Ca²⁺-ATPase, as shown by HRC overexpression in transgenic mouse hearts, which resulted in reduced SR Ca²⁺-uptake rates, cardiac remodeling and hypertrophy. In fact, in vitro generated evidence suggests that HRC directly interacts with SR Ca²⁺-ATPase2, supporting a dual role of HRC in Ca²⁺-homeostasis: regulation of both SR Ca²⁺-uptake and Ca²⁺-release. Furthermore, HRC plays an important role in myocyte differentiation and in antiapoptotic cardioprotection against ischemia/reperfusion induced cardiac injury. Interestingly, HRC has been linked with familiar cardiac conduction disease and an HRC polymorphism was shown to associate with malignant ventricular arrhythmias in the background of idiopathic dilated cardiomyopathy. This review summarizes studies, which have established the critical role of HRC in Ca²⁺-homeostasis, suggesting its importance in cardiac physiology and pathophysiology.     

Limitations of FKBP12.6-directed treatment strategies for maladaptive cardiac remodeling and heart failure

Sarcoplasmic reticulum (SR) calcium (Ca) leak can be reduced by enhancing FKBP12.6 binding to SR Ca release channels (RyR2) and expression of a “sticky” FKBP12.6^D37S mutant may correct reduced binding stoichiometry in RyR2 from failing hearts. Both calcium/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and protein kinase A (PKA) are activated in heart failure and promote SR Ca leak at RyR2. It is possible that FKBP12.6 dissociation from RyR2 may promote remodeling and that interventions to reassociate FKBP12.6 with RyR2 reflect a future therapeutic strategy. We created transgenic (TG) mice expressing FKBP12.6^D37S and tested their capacity to improve intracellular Ca handling and pathological remodeling in vivo. FKBP12.6^D37S TG mice were cross-bred with CaMKIIδc TG mice, which are known to exhibit pronounced RyR2 dysfunction and heart failure. We observed a significant improvement of post-rest Ca transients and a higher SR Ca content in FKBP12.6^D37S TG mice. In double-TG mice, a marked reduction of SR Ca spark frequency indicated reduced SR Ca leak but neither SR Ca transient amplitude, SR Ca content nor morphological or functional parameters improved in vivo. Likewise, FKBP12.6^D37S TG mice subjected to increased afterload after aortic banding exhibited higher SR Ca load but did not exhibit any improvement in hypertrophic growth or functional decline. Enhancement of FKBP12.6-RyR2 binding markedly reduced RyR2 Ca leak in CaMKIIδc-induced heart failure and in pressure overload. Our data suggest that activation of CaMKIIδc and pressure overload confer significant resistance towards approaches aiming at FKBP12.6-RyR2 reconstitution in heart failure and maladaptive remodeling, although RyR2 Ca leak can be reduced.     

CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2 + leak and the pathophysiological response to chronic β-adrenergic stimulation

Chronic activation of Ca^2 +/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca^2 + leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca^2 + leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Role of CaMKII in RyR leak, EC coupling and action potential duration: A computational model

During heart failure, the ability of the sarcoplasmic reticulum (SR) to store Ca²⁺ is severely impaired resulting in abnormal Ca²⁺ cycling and excitation-contraction (EC) coupling. Recently, it has been proposed that “leaky” ryanodine receptors (RyRs) contribute to diminished Ca²⁺ levels in the SR. Various groups have experimentally investigated the effects of RyR phosphorylation mediated by Ca²⁺/calmodulin-dependent kinase II (CaMKII) on RyR behavior. Some of these results are difficult to interpret since RyR gating is modulated by many external proteins and ions, including Ca²⁺. Here, we present a mathematical model representing CaMKII-RyR interaction in the canine ventricular myocyte. This is an extension of our previous model which characterized CaMKII phosphorylation of L-type Ca²⁺ channels (LCCs) in the cardiac dyad. In this model, it is assumed that upon phosphorylation, RyR Ca²⁺-sensitivity is increased. Individual RyR phosphorylation is modeled as a function of dyadic CaMKII activity, which is modulated by local Ca²⁺ levels. The model is constrained by experimental measurements of Ca²⁺ spark frequency and steady state RyR phosphorylation. It replicates steady state RyR (leak) fluxes in the range measured in experiments without the addition of a separate passive leak pathway. Simulation results suggest that under physiological conditions, CaMKII phosphorylation of LCCs ultimately has a greater effect on RyR flux as compared with RyR phosphorylation. We also show that phosphorylation of LCCs decreases EC coupling gain significantly and increases action potential duration. These results suggest that LCC phosphorylation sites may be a more effective target than RyR sites in modulating diastolic RyR flux.     

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr¹⁷ site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na⁺/Ca²⁺exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca²⁺-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca²⁺ loading and release (thapsigargin/dantrolene), mitochondrial Ca²⁺ uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr¹⁷ of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.     

Sarcoplasmic reticulum Ca2+ release in neonatal rat cardiac myocytes

In the neonatal mammalian heart, the role of ryanodine receptor (= Ca²⁺ release channel)-mediated sarcoplasmic reticulum (SR) Ca²⁺ release for excitation–contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes. Measurement of Ca²⁺ transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca²⁺ content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca²⁺ storage but unchanged Ca²⁺ signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation–contraction coupling in the developing mammalian heart.     

Improvement of Cardiac Sarcoplasmic Reticulum Calcium Cycling in Dogs with Heart Failure Following Long-Term Therapy with the <Emphasis Type="Italic">Acorn</Emphasis> Cardiac Support Device

Abnormal Ca²⁺-homeostasis is a hall-marked characteristic of the failing heart. In the normal myocardium, the sarcoplasmic reticulum (SR) is a principal organelle that controls intracellular Ca²⁺ concentration during the cardiac cycle. The SR consists of longitudinal and terminal cisternea regions. The former contains the Ca²⁺-ATPase pump or SERCA-2a whose function is to transport cytosolic Ca²⁺ into the lumen of the SR during diastole and whose activity is regulated by reversible phosphorylation of the endogenously SR-bound phospholamban (PLB). The SR's terminal cisternea region contains ryanodine-sensitive Ca²⁺-release channels (RR), the activity of which is regulated by direct and indirect reversible phosphorylation. These channels release the SR-stored Ca²⁺ during contraction. We have shown that in left ventricular (LV) myocardium from dogs with coronary microembolization-induced heart failure, ability of the SR to sequester and release Ca²⁺ during the cardiac cycles is impaired. This abnormality was associated with reduced expression (protein and mRNA) levels of Ca²⁺-ATPase, PLB, and reduced PLB phosphorylation. Long-term therapy with the Acorn Cardiac Support Device (CSD) is associated with restoration of the ability of the SR to sequester Ca²⁺. This improvement in SR function following chronic CSD therapy was due primarily to increased affinity of the SERCA-2a for calcium. The later was associated with (1) increased phosphorylation of PLB at serine 16 and threonine 17, (2) unchanged protein expression of PLB and (3) unchanged protein expression of SERCA-2a in LV myocardium of CSD-treated dogs compared to controls. This review summarizes our current understanding of the role of the CSD in modulating SR calcium cycling in an experimental canine model of chronic heart failure produced by multiple sequential intracoronary microembolizations.     

Reduced sarcoplasmic reticulum Ca2+ uptake and increased Na+–Ca2+ exchanger expression in left ventricle myocardium of dogs with progression of heart failure

Alteration of intracellular Ca²⁺ homeostasis in failing cardiomyocytes is associated with changes in regulatory proteins located in the sarcoplasmic reticulum (SR) and sarcolemma, which participate in Ca²⁺ fluxes across the membrane during the cardiac cycle. These regulatory proteins include Ca²⁺-ATPase (SERCA 2A), phospholamban (PLB), ryanodine-sensitive Ca²⁺ release channels (RR), and the sarcolemmal Na⁺–Ca²⁺ exchanger (NCX). Although their status is known in failed myocardium, it is poorly understood during the progression of heart failure (HF), particularly in large animals. We studied the left ventricular (LV) myocardium of six dogs with moderate HF and six with severe HF produced by multiple intracoronary microembolizations, compared with six normal dogs (NL). Oxalate-dependent SR Ca²⁺ uptake and expression of SERCA 2A, PLB, phosphorylated PLB at serine 16 (PLB-Ser) and threonine 17 (PLB-Thr), RR, and NCX were determined. Percent LV ejection fraction declined by 47% compared with NL (34.1% ± 1% vs 64% ± 2%) in dogs with moderate HF (HF-2W) 2 weeks after the last embolization and by 42% (20.5% ± 1% vs 34.1% ± 1%) in dogs with severe HF (HF-4M) at 4 months compared with HF-2W. Left ventricular pressure during isovolumic contraction (+dP/dt, mmHg/s) and relaxation (–dP/dt, mmHg/s) was significantly reduced in severe compared with moderate HF. Oxalate-dependent SR Ca²⁺ uptake (nmol ⁴⁵Ca²⁺ accumulated/min per milligram noncollagen protein) declined by 25% (21.3 ± 1 vs 28.5 ± 2) in HF-2W and 49% in HF-4M. Protein expression of SERCA 2A and PLB decreased by 67% and 35%, respectively, in HF-2W compared with NL, whereas SERCA 2A expression increased by 167% and PLB decreased by 40% in HF-4M compared with HF-2W. However, SERCA 2A protein was still significantly lower in HF-4M compared with NL. PLB-Ser and PLB-Thr increased significantly in HF-2W but decreased in HF-4M compared with NL. Similar changes in mRNA encoding PLB and SERCA 2A were observed in dogs with moderate and severe HF. The RR protein level declined in dogs with moderate and severe HF, whereas NCX protein did not change with moderate HF but increased with sever HF. These results suggest that the regulatory proteins responsible for Ca²⁺ uptake, Ca²⁺ release, and Na⁺–Ca²⁺ exchange are critically associated with the deterioration of LV function during the progression of HF.     

Intracellular mechanisms of specific beta-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure

β-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (α_2A/α_2C-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca²⁺]_i transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca²⁺]_i transient, carvedilol had no effect on peak [Ca²⁺]_i transient but also increased [Ca²⁺] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca²⁺ transients and the net balance of cardiac Ca²⁺ handling proteins while carvedilol preferentially improved cardiac redox state.     

Calmodulin kinase II inhibition disrupts cardiomyopathic effects of enhanced green fluorescent protein

Transgenic expression of enhanced green fluorescent protein (eGFP) in myocardium can result in cardiac dysfunction and cardiomyopathy, presumably through toxic effects that disrupt normal cellular signaling. The multifunctional Ca²⁺- and calmodulin-dependent protein kinase II (CaMKII) is widely expressed in myocardium and CaMKII activity is increased in human and animal models of cardiomyopathy, so we hypothesized that increased CaMKII activity is important for cardiomyopathy due to transgenic expression of eGFP. Here we report that cardiomyocyte-delimited eGFP over-expression causes increased CaMKII activity that predicts left ventricular dilation and dysfunction. On the other hand, transgenic co-expression of a CaMKII inhibitory peptide with eGFP prevents eGFP-mediated left ventricular dilation and dysfunction. These findings suggest that increased CaMKII activity is a critical pathological signal in transgenic cardiomyopathy due to eGFP over-expression.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Modulation of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> cycling in systolic and diastolic heart failure associated with aging

Hypertension, atherosclerosis, and resultant chronic heart failure (HF) reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. Thus, age per se is the major risk factor for cardiovascular disease. Changes in cardiac cell phenotype that occur with normal aging, as well as in HF associated with aging, include deficits in ß-adrenergic receptor (ß-AR) signaling, increased generation of reactive oxygen species (ROS), and altered excitation–contraction (EC) coupling that involves prolongation of the action potential (AP), intracellular Ca²⁺ (Ca _i ²⁺ ) transient and contraction, and blunted force- and relaxation-frequency responses. Evidence suggests that altered sarcoplasmic reticulum (SR) Ca²⁺ uptake, storage, and release play central role in these changes, which also involve sarcolemmal L-type Ca²⁺ channel (LCC), Na⁺–Ca²⁺ exchanger (NCX), and K⁺ channels. We review the age-associated changes in the expression and function of Ca²⁺ transporting proteins, and functional consequences of these changes at the cardiac myocyte and organ levels. We also review sexual dimorphism and self-renewal of the heart in the context of cardiac aging and HF.     

Mechanoelectric coupling and arrhythmogenesis in cardiomyocytes contracting under mechanical afterload in a 3D viscoelastic hydrogel

The heart pumps blood against the mechanical afterload from arterial resistance, and increased afterload may alter cardiac electrophysiology and contribute to life-threatening arrhythmias. However, the cellular and molecular mechanisms underlying mechanoelectric coupling in cardiomyocytes remain unclear. We developed an innovative patch-clamp-in-gel technology to embed cardiomyocytes in a three-dimensional (3D) viscoelastic hydrogel that imposes an afterload during regular myocyte contraction. Here, we investigated how afterload affects action potentials, ionic currents, intracellular Ca²⁺ transients, and cell contraction of adult rabbit ventricular cardiomyocytes. We found that afterload prolonged action potential duration (APD), increased transient outward K⁺ current, decreased inward rectifier K⁺ current, and increased L-type Ca²⁺ current. Increased Ca²⁺ entry caused enhanced Ca²⁺ transients and contractility. Moreover, elevated afterload led to discordant alternans in APD and Ca²⁺ transient. Ca²⁺ alternans persisted under action potential clamp, indicating that the alternans was Ca²⁺ dependent. Furthermore, all these afterload effects were significantly attenuated by inhibiting nitric oxide synthase 1 (NOS1). Taken together, our data reveal a mechano-chemo-electrotransduction (MCET) mechanism that acutely transduces afterload through NOS1–nitric oxide signaling to modulate the action potential, Ca²⁺ transient, and contractility. The MCET pathway provides a feedback loop in excitation–Ca²⁺ signaling–contraction coupling, enabling autoregulation of contractility in cardiomyocytes in response to afterload. This MCET mechanism is integral to the individual cardiomyocyte (and thus the heart) to intrinsically enhance its contractility in response to the load against which it has to do work. While this MCET is largely compensatory for physiological load changes, it may also increase susceptibility to arrhythmias under excessive pathological loading.

Biological cells are capable of sensing and responding to mechanical forces. In every heartbeat, cardiomyocytes experience large dynamic changes in mechanical forces during diastolic filling (preload, which exerts mechanical strain or stretch on the cell) and systolic contraction against resistance (afterload, which exerts mechanical stress on the cell). In addition to neurohormonal regulation, the heart has intrinsic abilities to regulate the contractile force in response to preload [via the Frank–Starling mechanism (1–3)] or afterload [via the Anrep effect (4, 5)] in order to maintain cardiac output. Changes in mechanical load also regulate electrical activity, referred to as mechano-electric coupling [MEC (6, 7)], which may contribute to cardiac arrhythmias (8, 9). Several ion channels have been found directly mechano-gated or modulated by the mechanical load–induced signaling pathways (10). However, more than 100 y after Bainbridge’s discovery of MEC (11), the exact ionic mechanisms by which mechanical load modulates the electrical activity of the cardiomyocyte remain elusive.We recently developed a patch-clamp-in-gel technique (12) based on our cell-in-gel system in which isolated cardiomyocytes are embedded in a three-dimensional (3D) viscoelastic hydrogel polymer matrix to apply afterload during cell contraction and investigate the cellular and molecular consequences of increased afterload (13, 14). This approach to apply afterload (mechanical stress) on the cardiomyocyte is profoundly different from previous studies using osmotic swelling, inflation, or uniaxial stretch to apply preload (mechanical strain) on the cardiomyocyte (15). Moreover, the cell-in-gel system imposes afterload during cyclic cardiomyocyte contraction as opposed to a static preload stretch used in most previous studies, with a few exceptions (16). To our knowledge, no data has been available on the ionic mechanisms of MEC under any kind of cyclic mechanical load during cardiomyocyte contraction. Hence, we aimed to study the afterload effects on regulating ion channels and action potential (AP) in cardiomyocytes during contraction in the beat-by-beat cardiac cycle.Our previous study showed that mouse ventricular cardiomyocytes contracting in a 3D viscoelastic hydrogel exhibited increased intracellular Ca²⁺ transients and increased diastolic Ca²⁺ sparks, with both dependent on localized nitric oxide synthase 1 (NOS1) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) signaling (13). In addition to mechanical loading, pathological changes in Ca²⁺ handling and nitric oxide (NO) signaling have also been linked to cardiac arrhythmias (17, 18). Therefore, we investigated the ionic mechanisms of afterload-induced MEC in rabbit (rather than mouse) ventricular myocytes in which the electrophysiology and Ca²⁺ handling properties resemble that of the human heart. We tested the hypothesis that mechanical afterload affects ion channels and Ca²⁺-handling molecules to regulate the AP, Ca²⁺ transient, and contractility in cardiomyocytes. Here, we used the patch-clamp-in-gel methodology to perform current-clamp, voltage-clamp, and AP-clamp experiments while the cardiomyocyte is contracting under afterload in a 3D viscoelastic hydrogel. We found that the afterload on a contracting cardiomyocyte regulated multiple ionic currents, including L-type Ca²⁺ current (I_Ca,L), transient outward K⁺ current (I_to), inward rectifier K⁺ current (I_K1), and a mechanosensitive current inhibited by GsMTx-4 (19), leading to prolonged AP duration (APD) and enhanced Ca²⁺ transient and contractility. However, this afterload also promoted discordant alternans in APD and Ca²⁺ transient, increasing the susceptibility to cardiac arrhythmias. Furthermore, we found that the afterload effects were critically mediated by the localized NOS1–NO signaling.     

The human CASQ2 mutation K206N is associated with hyperglycosylation and altered cellular calcium handling

Mutations in the human cardiac calsequestrin gene (CASQ2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT-2). This inherited disorder is characterized by life-threatening arrhythmias induced by physical and emotional stress in young patients. Here we identified a novel heterozygous missense mutation (K206N) in the CASQ2 gene in a symptomatic family in which one member died of cardiac arrest. The functional properties of CSQ^K206N were investigated in comparison to the wild-type form of CASQ2 (CSQ^WT) by expression in eukaryotic cell lines and neonatal mouse myocytes. The mutation created an additional N-glycosylation site resulting in a higher molecular weight form of the recombinant protein on immunoblots. The mutation reduced the Ca²⁺ binding capacity of the protein and exhibited an altered aggregation state. Consistently, CSQ^K206N-expressing myocytes exhibited an impaired response to caffeine administration, suggesting a lower Ca²⁺ load of the sarcoplasmic reticulum (SR). The interaction of the mutated CSQ with triadin and the protein levels of the ryanodine receptor were unchanged but the maximal specific [³H]ryanodine binding was increased in CSQ^K206N-expressing myocytes, suggesting a higher opening state of the SR Ca²⁺ release channel. Myocytes with expression of CSQ^K206N showed a higher rate of spontaneous SR Ca²⁺ releases under basal conditions and after β-adrenergic stimulation. We conclude that CSQ^K206N caused a reduced Ca²⁺ binding leading to an abnormal regulation of intracellular Ca²⁺ in myocytes. This may then contribute to the increased propensity to trigger spontaneous Ca²⁺ transients in CSQ^K206N-expressing myocytes.