Adult Ventricular Cardiac Myocytes Research Articles

Internalized β2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling

Chronic overstimulation of Gs-coupled b-adrenergic receptor (b-AR) signaling by catecholamines, induces pathological cardiac hypertrophy and ultimately heart failure. b1-ARs and b2-ARs are the two major subtypes of b-ARs present in the human heart and mediate the activity of sympathetic nervous system. It is known that b1-ARs and b2-ARs elicit significantly different or even opposite effects on cardiac functions such as contractility and heart failure, though they share overall sequence and structural homology, and both stimulate cAMP production. The molecular mechanisms underlying these distinct biological effects have not been fully elucidated. Previously, our laboratory demonstrated that a cAMP/EPAC/phospholipase Ce (PLCe)-mediated signaling pathway at the Golgi apparatus is important for regulation of cardiac hypertrophy. We also showed that this pathway is selectively stimulated by activation of b1-ARs localized at Golgi apparatus but not those at the plasma membrane (PM). Access to intracellular b1-ARs by catecholamines was mediated by the organic cation transporters (OCTs), and blockade of OCT3 inhibited catecholamine stimulated cardiomyocyte hypertrophy. In my current study using adult ventricular cardiac myocytes, I found that the activation of endosomal b2-ARs opposes stimulation of the prohypertrophic EPAC/PLCe pathway at the Golgi apparatus by either Angiotensin II (ATII), or the activation of Golgi localized b1-ARs by Dobutamine (Dob). b2-AR-dependent inhibition is at the level of PLCe since activation of b2-AR with salmeterol (Sal) blocks direct activation of EPAC/PLCe by the EPAC selective cAMP analog 8-(4-chlorophenylthio)-2′- O-methyl-cAMP (CPTOMe)-AM, and blocks PLCe activation by ATII which activates PLCe through a pathway that does not rely on EPAC. Blockade of internalization of b2-ARs with Dyngo-4A abolished this inhibitory signaling. Downstream of b2-ARs, I found that b2-AR-dependent PLCe inhibition is not mediated by PKA. Rather, blockade of Gi with PTX, or Gbg with gallein, inhibited the ability of b2-ARs to block Golgi PLCe activity. Additionally, inhibition of ERK activation by PD0325901 abolished b2-AR-mediated inhibition of Golgi PLCe activity. This supports a model where b2ARs internalized from the plasma membrane activate Gi releasing Gbg subunits leading to ERK activation and inhibition of PI hydrolysis at the Golgi apparatus, thereby inhibiting hypertrophic signaling by PLCe. In addition, we also showed PKD and HDAC phosphorylation downstream of PLCe signaling are significantly attenuated by b2ARs activation in mouse hearts. Consistent with the results from mice, b2ARs activation by Sal blunted cellular hypertrophy induced by b1-ARs activation by Dob in vitro measured by cell area and the expression of the hypertrophic marker, atrial natriuretic factor (ANF). Importantly, inhibition endosomal Gbg using a highly selective Gbg inhibitor (GRK2CT) fused to an endosomal targeting sequence (FYVE domain) prevent Sal mediated protective signaling in hypertrophy measurements in vitro. This indicates the inhibitory signaling downstream of b2ARs is indeed driven by endosomal Gbg. This study reveals a novel potential mechanism for b2-AR antagonism of the Epac/PLCe pathway that may contribute to the known protective effects of b2AR signaling on the development of heart failure. Elucidation of a mechanism for the anti-hypertrophic versus hypertrophic signaling balance from b1-ARs and b2-ARs gives critical insights into the development of new strategies for treatment of heart failure by targeting bAR subtypes. This work is supported by grant R35 GM127303-01 (A.V.S.). Preprint: https://www.biorxiv.org/content/10.1101/2023.06.07.544153v1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Restricted Diffusion of cAMP is Mediated by Mitochondrial Localized PKA Buffering in Living Cells

Different G-protein coupled receptors can elicit unique functional responses by selectively utilizing specific subsets of spatially restricted cAMP signaling domains. Previous studies have concluded that this can only be achieved if the rate of cAMP movement within cells is much slower than the rate of free diffusion. However, this prediction has not been demonstrated experimentally. In this study, we directly test the hypothesis that the cytoplasmic diffusion of cAMP is indeed slow and then address the long standing question of what regulates its movement. We used Raster Image Correlation Spectroscopy (RICS) to directly measure the diffusion coefficients of various molecules in intact HEK 293 cells and adult ventricular cardiac myocytes to evaluate the effect of particle size and cell morphology on cAMP movement. Fluorescein and pharos-450 (φ450), fluorescent molecules similar in size to cAMP, exhibited diffusion coefficients 3-4 fold slower than their expected rates of free diffusion. The diffusion coefficient of φ450-labeled cAMP (φ450-cAMP) was >6 fold slower than fluorescein or φ450 free dye alone. This extremely slow rate of diffusion could not be explained by its size. In cardiac myocytes, φ450-cAMP co-localized with the type II regulatory (RII) subunit of protein kinase A (PKA). In addition, disruption of RII subunit anchoring to A kinase anchoring proteins (AKAPs) significantly increased the diffusion coefficients of both RII subunits and φ450-cAMP. Furthermore, RII subunits and φ450-cAMP co-localized with mitochondria. These results demonstrate that the cytosolic movement of cAMP is indeed much slower than the predicted rate of free diffusion and that this is due to interactions with PKA regulatory subunits associated specifically with mitochondria. These findings have important implications with respect to cAMP signaling in all cell types.

Functional Expression and Subcellular Localization of F-Channels in Human Ventricular and hESC-Derived Cardiomyocytes

Caveolae are specialized lipid rafts in the plasma membrane responsible for the interaction, sublocalization and function of proteins and ion channels. The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) genes encode for the alpha subunit of f-channel present in cardiac pacemaker cells, in the adult and developing cardiac myocytes (CM). HCN4 is the predominant isoform in the sinoatrial node and, in the rabbit, experimental evidence indicate that it localizes into membrane caveolae, where caveolin-3 (Cav3)-channel interaction regulates current properties and autonomic modulation. HCN4 is abundant in undifferentiated human embryonic stem cells (hESC) and immature hESC-derived CM (hESC-CM). To date, no information is available on i) developmental changes of HCN4 channel localization and function in human CM ii) the relationship with HCN channel expression/function in adult CM.Confocal analysis showed that HCN4 and Cav3 colocalize in adult human ventricular CM. In the same cells, f-current was consistently recorded upon hyperpolarization (70% cells), with a voltage of half maximal activation (Vh) of −94 mV. Protein and mRNA for Cav3 were not detected in undifferentiated hESC, but expression increased during maturation of hESC-CM. Oppositely, HCN4 was highly expressed in hESC and early hESC-CM, but a 5-fold decrease in mRNA levels occurred in late hESC-CM. In these cells, HCN4 appeared to be associated with Cav3. Activation properties of f-current recorded in late hESC-CM resembled those measured in adult ventricular CM (Vh=-93 mV). Current activation was faster and occurred at more positive potentials in hESC and early CM. In conclusion, cardiac maturation is associated with the recruitment of HCN4 channel and CAV3 into membrane lipid rafts, suggesting that sub-cellular localization of f-channel in lipid rafts is a fundamental step during cardiac maturation.

L-Type Calcium Channel C Terminus Autoregulates Transcription

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. The guiding hypothesis of this study is that a carboxyl-terminal cleavage product of the cardiac L-type calcium channel (Ca(V)1.2) autoregulates expression. First, we confirmed that the Ca(V)1.2 C terminus (CCt) is cleaved in murine cardiac myocytes from mature and developing ventricle. Overexpression of full-length CCt caused a 34+/-8% decrease of Ca(V)1.2 promoter activity, and truncated CCt caused an 80+/-3% decrease of Ca(V)1.2 promoter (n=12). The full-length CCt distributes into cytosol and nucleus. A deletion mutant of CCt has a greater relative affinity for the nucleus than full-length CCt, and this is consistent with increased repression of Ca(V)1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed that CCt interacts with the Ca(V)1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing Nkx2.5/Mef2, C/EBp, and a cis regulatory module. The next hypothesis tested was that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We explored whether fetal cardiac myocyte Ca(V)1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of Ca(V)1.2 promoter, protein, and L-type current (I(Ca,L)) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased Ca(V)1.2 promoter activity, mRNA, and I(Ca,L). Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analyses, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca(V)1.2, autoregulates Ca(V)1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca(V)1.2 links Ca handling to nuclear signaling.

Abstract 1875: L-type Calcium Channel C-terminus Suppresses L-type Calcium Channel Transcription in Cardiomyocytes

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. In this study we test the hypothesis that a mobile carboxyl-terminal cleavage product (CCt) of the cardiac L-type calcium channel (Ca V 1.2) auto-regulates expression. Our ongoing in vivo studies showed that persistent Ca V 1.2 block induced an up-regulation of Ca V 1.2 mRNA, protein and I Ca,L . In the present study we test the hypothesis that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We created eGFP-CCt fusion constructs to explore CCt regulation of the Ca V 1.2 promoter, and to report sub-cellular localization of CCt in cardiac myocytes. Developing cardiac myocytes were cultured in 10% FBS. Over-expression of native, full-length CCt causes a 34 ± 8% decrease of Ca V 1.2 promoter activity, and truncated CCt caused 80 ± 3% decrease (n = 12). Wild-type CCt distributes into cytosol and nucleus, with a greater presence in the nucleus than cytosol. A deletion mutant of CCt was also tested. Truncated CCt has a greater relative affinity for the nucleus than wild-type CCt. This is consistent with increased suppression of Ca V 1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation (ChIP) assay showed that CCt interacts with the Ca V 1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing NKX2.5/MEF2, C/EBp, and a cis regulatory module identified in silico . We next explored whether fetal cardiac myocyte Ca V 1.2 was regulated by serum in vitro . We tested ANF promoter activity as a positive control, and measured serum-response, versus serum-withdrawal on Ca V 1.2 promoter, mRNA, protein, and L-type current (I Ca,L ) from fetal mouse ventricular myocytes. Serum increased ANF promoter activity and cell size. Serum withdrawal increased Ca V 1.2 promoter activity, Ca V 1.2 protein, and I Ca,L . Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analysis, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca V 1.2 auto-regulates Ca V 1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca V 1.2 links Ca handling to nuclear signaling.

Dichotomy of Ca2+ in the heart: contraction versus intracellular signaling

Ca2+ plays a pivotal role in both excitation-contraction coupling (ECC) and activation of Ca2+-dependent signaling pathways. One of the remaining questions in cardiac biology is how Ca2+-dependent signaling pathways are regulated under conditions of continual Ca2+ transients that mediate cardiac contraction during each heartbeat. Ca2+-calmodulin-dependent protein kinase II (CaMKII) activation and its ability to regulate histone deacetylase 5 (HDAC5) nuclear shuttling represent a critical Ca2+-dependent signaling circuit for controlling cardiac hypertrophy and heart failure, yet the mechanism of activation by Ca2+ is not known. In this issue of the JCI, Wu et al. convincingly demonstrate that the inositol 1,4,5-trisphosphate receptor (InsP3R) is involved in local control of Ca2+ for activating CaMKII in the nuclear envelope of adult ventricular cardiac myocytes (see the related article beginning on page 675). The overall paradigm that is demonstrated is the best example of a molecular mechanism whereby signaling is directly regulated by a local Ca2+ pool that is disparate or geometrically insensitive to cytosolic Ca2+ underlying each contractile cycle.

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo.

Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes - E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field.

Mode-dependent inhibition by quinidine of Na+-Ca2+ exchanger current from guinea-pig isolated ventricular myocytes

1. The aim of the present study was to determine whether or not the class Ia anti-arrhythmic agent quinidine modulates ionic current (INaCa) generated by the sarcolemmal Na+-Ca2+ exchanger of adult ventricular cardiac myocytes. 2. Selective whole-cell voltage-clamp recordings of INaCa were made from guinea-pig ventricular myocytes, with major interfering currents blocked. The INaCa was measured as the ionic current sensitive to 10 mmol/L external Ni2+ during a descending voltage ramp protocol. 3. The effects of quinidine concentrations in the range 10-100 micromol/L were studied. Quinidine produced a concentration-dependent partial blockade of outward INaCa, generated by reverse-mode exchange. At +60 mV, 100 micromol/L quinidine blocked INaCa by 33.0 +/- 4.1% (mean+/-SEM; n = 4). This was the maximal concentration that we were able to test, because concentrations of quinidine higher than 100 micromol/L were found to be toxic to cells under our conditions. The drug did not produce any significant inhibition of inward INaCa (generated by forward-mode exchange). 4. The Ni2+-insensitive residual current was not significantly altered by quinidine at any membrane potential, confirming that the inhibitory effects of quinidine we observed could be attributed to an action on the Na+-Ca2+ exchanger. 5. For the purpose of comparison, quinidine was tested against L-type Ca current (ICa,L). It blocked peak ICa,L at 0 mV, with an IC50 of 14.9 +/- 1.5 micromol/L. Thus, quinidine was less potent against the exchanger than against ICa,L. 6. Our data suggest that quinidine preferentially inhibits the Naout/Cain mode of exchanger function. We conclude that this drug is a weak inhibitor of ventricular INaCa and that the inhibitory effect is mode dependent.

Differential effects of carvedilol and metoprolol on isoprenaline-induced changes in β-adrenoceptor density and systolic function in rat cardiac myocytes

beta-Blockers improve cardiac function and survival in heart failure patients. The underlying mechanisms are not completely elucidated. Differences between agents might be important for the development of more specific therapeutical approaches. This study investigated whether metoprolol or carvedilol alter beta-adrenergic signaling differently. beta-Adrenoceptor density and systolic function were determined in rat adult ventricular cardiac myocytes. 12 h isoprenaline-treatment (Iso, 1 micromol/l) reduced beta-adrenoceptor density by 33% (P<0.01). The effect was abolished by incubation with isoprenaline plus metoprolol (3 micromol/l), but was more pronounced after coincubation with carvedilol (0.003 micromol/l, P<0.05 Carv vs. Iso). Metoprolol alone had no effect on beta-adrenoceptor density, but carvedilol induced a decrease in receptor density even in absence of isoprenaline (P<0.05 Carv vs. ctr.). The isoprenaline (0.0003-10 micromol/l) induced concentration-dependent increase in myocyte shortening was blunted after 12 h preincubation with Iso (1 micromol/l, P<0.001). This reduction was abolished or partly prevented by coincubation with metoprolol or carvedilol, respectively. Carvedilol decreased the number of receptors which had to be occupied by isoprenaline in order to obtain 50% and 90% increase in myocyte cell shortening. Comparison of guanine nucleotide-dependent binding characteristics of isoprenaline, carvedilol and metoprolol revealed beta-receptor agonist like binding characteristics for carvedilol, but antagonist like binding characteristics for metoprolol. Metoprolol but not carvedilol prevents isoprenaline-induced downregulation of myocyte beta-adrenoceptors. The difference might be due to specific binding properties of the beta-blockers. Restoration of isoprenaline responsiveness by carvedilol might be due to improved coupling of beta-receptors to postreceptor effects.

Endothelin stimulates multiple responses in isolated adult ventricular cardiac myocytes.

We have examined the responses to endothelin (ET) in isolated adult cardiac myocytes from both rodent and feline species to assess whether endothelin may have a role in the induction or mediation of cardiac hypertrophy in the adult animal. We have evidence that ET acts by more than one mechanism to promote cell-signaling events believed important in growth regulation. In isolated adult cardiac ventricular myocytes labeled overnight with [3H]inositol, endothelin (ET) promoted a two- to fourfold increase in the accumulation of inositol polyphosphates in a dose-dependent manner with an half-maximal effective concentration (EC50) of approximately 5 nM. In contrast, picomolar concentrations of ET promoted an increase in both the extent and velocity of sarcomere shortening in electrically stimulated myocytes. Pretreatment of cells with pertussis toxin had no effect on the ET-stimulated phosphoinositide hydrolysis, but blocked the ET-stimulated positive inotropic effect. In addition to these early responses to ET, results obtained by Northern blot analysis demonstrate that exposure of isolated cardiac myocytes to 100 nM ET promoted the expression of c-fos and c-zif in both mammalian species. These data demonstrate that ET stimulates multiple cell-signaling pathways in adult mammalian cardiac myocytes. A paracrine mechanism of regulation of adult myocardium is suggested.