α-1 Subunit

If the main effects of β-AR stimulation on voltage dependence and amplitude of the I_Ca,L and their consequences for the fight-or-flight response are well documented in the literature, the molecular events that mediate the increase of Ca_V1.2 activity during a sympathetic stimulation remain elusive; this is due to the difficulty of reconstituting such modulation in heterologous overexpression systems. The α_1C-subunit of Ca_V1.2 channels exhibits multiple potential PKA phosphorylation sites in the N- and the C-terminal regions (Figure 37-3).² Despite the fact that α_1C is a substrate for phosphorylation by PKA in vitro,^16,17 several attempts to mimic the adrenergic stimulation of Ca_V1.2 channels in expression systems have failed.^18–20 This failure led to the hypothesis that at least one missing link in heterologous systems would preclude the reconstitution of such modulation of overexpressed Ca_V1.2 channels. One of these links could be an A-kinase-anchoring protein (AKAP) that allows the cAMP modulation and the PKA phosphorylation of serine 1928 in the C-terminus of Ca_V1.2 channels in HEK293 cells.²¹ A conserved leucine zipper motif in the C-terminus of Ca_V1.2 identified in native cardiac cells directly anchors a low molecular weight AKAP15-PKA complex to ensure a fast and efficient β-adrenergic modulation of I_Ca,L (see Figure 37-3),²² allowing its phosphorylation at serine 1928 when β-ARs are stimulated.²³ Surprisingly, the C-terminal part of rabbit Ca_V1.2 undergoes proteolytic processing by calpain at residue 1821, leaving two size forms of the α_1C-subunit of these channels expressed in cardiac cells,¹⁷ whereas this distal 37 to 50-kDa peptide contains the serine 1928 phosphorylated by PKA and the binding site for AKAP15. In fact, this fragment constitutes a potent autoinhibitory domain that covalently associates with the proximal C-terminal part of α_1C, reducing its open probability and shifting the voltage dependence of activation to depolarized potentials.²⁴ The role of serine 1928 in I_Ca,L upregulation by β-AR has since been challenged. A first study showed that a DHP-resistant Ca_V1.2 channels mutated at position 1928 (S1928A) displays an unaltered response to the β-AR agonist isoproterenol when overexpressed in isolated ventricular myocytes.²⁵ Moreover, the generation of a S1928A knock-in mouse model confirmed that the phosphorylation of this residue by PKA is not required for the functional effects of β-AR stimulation on Ca_V1.2 in cardiac cells, because these mice exhibit an essentially conserved response of I_Ca,L to isoproterenol and typical chronotropic and inotropic responses to β-AR stimulation.²⁶ Therefore, although phosphorylation of serine 1928 within the C-terminus of Ca_V1.2 definitely occurs when β-ARs are stimulated,^17,21,23 it does not correlate with the functional effects observed on I_Ca,L. As a result, PKA would phosphorylate other PKA sites within the channel or another binding partner to mediate the increase of its activity.

β-Subunit

Three serines (S459, S478, and S479) of the cardiac the Ca_Vβ_2a subunit were identified as putative PKA phosphorylation sites.²⁷ Coexpression of this subunit with a Ca_V1.2 lacking the C-terminal part including the serine 1928 in TsA-201 cells allowed an upregulation of the current by cAMP/PKA pathway, whereas expression of mutated Ca_Vβ_2a at serine 478 and 479, but not at position 459, was unable to do so.²⁸ The authors concluded that the Ca_Vβ_2a ancillary subunit was the main target for PKA to mediate the β–AR stimulation of I_Ca,L; however, this conclusion has been questioned by experiments realized in a more physiologic context. Overexpression of a Ca_Vβ_2a construct mutated for its PKA phosphorylation sites in ventricular cardiac cells did not prevent the cAMP/PKA modulation of I_Ca,L.¹⁴ Nonetheless, the Ca_Vβ₂ subunit can associate with the giant cytoskeletal protein of 700 kDa named ahnak, which emerged as an important player in the β-AR regulation of cardiac I_Ca,L (see Figure 37-3). Through its interaction with Ca_Vβ₂, ahnak would serve as a brake on I_Ca,L that would be relieved by PKA phosphorylation of the ancillary subunit and the cytoskeletal protein.²⁹ Interestingly, ahnak polymorphism occurs, and the genetic variant generated interferes with the β-AR stimulation of I_Ca,L by reducing the Ca_Vβ₂ interaction with ahnak.²⁹ However, if the involvement of the Ca_Vβ or other binding partners such as ahnak cannot be ruled out completely, recent studies have reaffirmed the importance of the proteolytically cleaved distal C-terminus of Ca_V1.2 for its upregulation upon β-AR stimulation. In addition, a new model for the molecular basis of β-AR stimulation of Ca_V1.2 has been proposed.³⁰ Overexpression in TsA-201 cells of a truncated Ca_V1.2 channel at position A1800 (the site of in vivo proteolytic cleavage previously determined for skeletal muscle Ca_V1.1 channel³¹) with α₂δ₁ and Ca_Vβ_1b and the distal C-terminal part of the channel (peptide from 1821-2171) produced a functional channel that is autoinhibited. Two presumed sites for PKA phosphorylation were identified upstream from the proteolytic site—a serine at position 1700 and a threonine 1704 at the interface of the distal and the proximal C-terminal parts. Although a mutation of S1928 confirmed that its phosphorylation is not required for the increase of the current, substitution of T1704 and S1700 for alanines revealed that phosphorylation of T1704 is required for basal Ca_V1.2 channel activity, whereas S1700 is crucial for its cAMP/PKA-induced upregulation. In this scheme, the AKAP15 is still required to coordinate the PKA phosphorylation of S1700 that disrupts the interaction of the noncovalently distal C-terminus, thus relieving the inhibition of the channel.³⁰ This assumption is reinforced by the fact that mice expressing a Ca_V1.2 channel deleted for the distal C-terminus display altered cAMP/PKA regulation accompanied with reduced expression of AKAP15.³²

Receptor Specificity

Although only three types of β-adrenergic receptors (β-ARs) have been cloned (β₁-, β₂-, and β₃-ARs), the effect of catecholamines in the human heart is generally attributed to β₁– and β₂-ARs. β₁– and β₂-ARs are highly homologous receptors and are both positively coupled to AC/cAMP/PKA cascade, I_Ca,L and cardiac performance³³; however, they exert opposite effects on hypertrophy^38,39 and apoptosis,^40,41 and their respective contribution varies significantly depending on the cardiac tissue, pathophysiologic state, age, or developmental stage.⁴² Part of the difference is because β₂-ARs couple not only to G_αs but also to G_αi proteins, and this confines the cAMP-dependent signal to the membrane compartment and to activation of the LTCCs.⁴³ Another difference between the two β-AR subtypes is their location at the cell surface, with β₂-ARs present in the caveolae/lipid rafts^44,45 of the transverse-tubular structure⁴⁶ and β₁-ARs distributed throughout both caveolae–lipid rafts and nonlipid raft membrane domains⁴⁴ and in both plasma and T-tubular membranes.⁴⁶ Accordingly, the β₂-AR downstream activation of I_Ca,L is sensitive to disruption of caveolae by cholesterol depletion, whereas the β₁-AR stimulatory effect is not.⁴⁷ Therefore, β₂-AR stimulation exerts a local activation of LTCCs, whereas β₁-AR stimulation leads to activation of LTCCs in the distance.^43,48,49

The β₃-AR differs from β₁-and β₂-AR subtypes in its molecular structure and pharmacologic functions.⁵⁰ Expression of β₃-AR was demonstrated in human myocardium at the mRNA^51,52 and protein levels.^52–55 Interestingly, β₃-AR activation produces a negative inotropic effect in human endomyocardial biopsies from transplanted hearts^50,51 and in left ventricular samples from failing and nonfailing explanted hearts^50,54 that is due to activation of the NO/cGMP pathway, but increases I_Ca,L and contractility in human atrial tissue via the cAMP/PKA pathway (Figure 37-4).⁵⁶ This effect is reminiscent of the contractile effects of the serotonin 5-HT₄ receptors,⁵⁷ which are also coupled to increases in force of contraction⁵⁸ or I_Ca,L⁵⁹ in atria but not in healthy ventricles.

Role of A-Kinase-Anchoring Proteins

Cyclic AMP signaling components are organized into multiprotein complexes, an arrangement that increases both efficiency and specificity of the transduction cascade. AKAPs have an essential role in these arrangements. AKAPs form a large family of proteins comprising more than 50 members whose primary function is to anchor PKA in the vicinity of its substrates, thus ensuring the preferential phosphorylation of a limited number of targets.60,61 As discussed earlier, AKAP15 (also called AKAP 7 or AKAP15/18) is the main AKAP controlling the PKA phosphorylation of LTCCs. However, in a recent study, Ca_V1.2 phosphorylation and β-AR stimulation of I_Ca,L was found to be unchanged in mice in which the gene encoding this protein was inactivated, suggesting that PKA is anchored by a different protein to the channel.⁶² Furthermore, AKAP5 was found to target ACs, PKA and phosphatases within caveolae to allow specific PKA phosphorylation of the subpopulation of channels present in this compartment upon β-AR stimulation.⁶³ Although AKAPs share in common their ability to bind PKA, they are remarkably diverse scaffold proteins. Within each signalosome, AKAPs couple PKA to different substrates, enhancing the rate and fidelity of their phosphorylation by the kinase. Importantly, AKAPs not only bind PKA but act as scaffold proteins for other signaling components, including phosphatases 1 and 2,^64,65 Epac,⁶⁶ adenylyl cyclases,^67,68 and PDEs.⁶⁹ Recently, it has been demonstrated that the phosphoinositide 3-kinase p110γ, that was shown recently tether PKA,⁷⁰ orchestrates multiprotein complexes including different PDEs to control cardiac Ca_V1.2 phosphorylation during β-AR stimulation.⁷¹ The combination of PDEs and phosphatases present in individual AKAP complexes will affect the duration, amplitude, and spatial extent of cAMP/PKA signaling. Thus, by bringing together different combinations of upstream and downstream signaling molecules, AKAPs provide the architectural infrastructure for specialization of the cAMP signaling network.^61,66,72

Role of Phosphodiesterases

Localized cAMP signals can be generated by the interplay between discrete cAMP production sites and restricted diffusion within the cytoplasm. Restricted diffusion of cAMP can be achieved by several means. A first possibility is that physical barriers are created by specialized membrane structures within the cytoplasm. This method was initially proposed to explain the differences in cAMP concentration elicited by PGE₁ at the plasma membrane and in the bulk cytosol of HEK293 cells, although an experimental proof that this actually occurs is still lacking.⁷³ Another important mechanism that limits cAMP diffusion is cAMP degradation by PDEs, which appears to be critical to the formation of dynamic microdomains that confer specificity of the response.^35,60

Cardiac cAMP PDEs degrading belong to five families (PDE1 to PDE4 and PDE8) that can be distinguished by distinct enzymatic properties and pharmacology.⁷⁴ Among these families various enzymes were shown to degrade cAMP to allow a fine tuning of I_Ca,L regulation by PKA in the heart. Although PDE2 is not highly expressed in cardiomyocytes, it controls LTCC activity in various species, including human atrial myocytes.^75–78 This enzyme is activated by cGMP, and stimulation of guanylyl cyclase strongly decreases local cAMP levels controlling I_Ca,L with only modest effects on its global concentration, suggesting the existence of a cAMP microdomain including β-AR and LTCCs under tight control of PDE2.⁷⁹ Contrary to PDE2, PDE3 is inhibited by cGMP; this explains in part why cGMP at low concentration can also increase basal I_Ca,L as shown in human atrial myocytes.^78,80 In rodents, PDE3 and PDE4 are the major contributors to the total cAMP-hydrolytic activity^34,81 and PDE4 is dominant to modulate β-AR regulation of cAMP levels.^49,82–84 Multiple PDE4 variants associate with β-ARs,^85–87 RyR2,⁸⁸ SERCA2,^89,90 I_Ca,L,⁹¹ and I_Ks⁷² to exert local control of ECC. In larger mammals, PDE3 activity is dominant in microsomal fractions,^92–94 and PDE3 inhibitors exert a potent positive inotropic effect.⁹⁵ Selective inhibition of PDE3 with milrinone has been shown to improve cardiac contractility in patients with congestive heart failure.⁹⁶ The role of PDE4 is less well defined, but evidence is emerging that PDE4 could also have an important role in these species. In the canine heart, a large PDE4 activity is found in the cytoplasm,⁹² but PDE4 is also present in microsomal fractions, where it accounts for approximately 20% of the activity.⁹³ Recent studies have indicated that PDE4 is expressed in the human ventricle where, similar to rodents, it associates with β-ARs, RyR2, and phospholamban.^81,88 Moreover, PDE4 is the main PDE modulating LTCC activity in rodent cardiomyocytes,^77,84 and PDE4 was recently shown to control I_Ca,L, ECC and arrhythmias in the human atrium.⁹⁷

Early evidence of the contribution of PDEs to intracellular cyclic nucleotide compartmentation was obtained by comparing the effects of the nonselective β-AR agonist Iso, or the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the PDE3 inhibitor milrinone in guinea pig perfused hearts. Whereas each of these treatments increased intracellular cAMP and produced positive inotropic and lusitropic effects, differences in the phosphorylation pattern of PLB, TnI, and MyBP-C by PKA were observed.⁹⁸ These results were attributed to a functional cellular compartmentation of cAMP and PKA substrates owing to a different expression of PDEs at the membrane and in the cytosol.⁹² In canine ventricular myocytes, an increase in particulate but not total cAMP correlated to an increase in Ca²⁺ transient amplitude and decay kinetics.⁹⁹ In response to β-AR stimulation, approximately 45% of the total cAMP was found in the particulate fraction, but this fraction declined to less than 20% when IBMX was added to Iso, although cAMP production was up to threefold to fourfold greater. These results show that cAMP-PDEs reside predominantly in the cytoplasm, where they prevent excessive cAMP accumulation upon β-AR activation. Thus, PDEs appear to be important to maintain the specificity of the β-AR response by limiting the amount of cAMP diffusing from membrane to cytoplasm. Similar results were obtained when studying the effect of PDE inhibition on I_Ca,L regulation by local application of Iso in frog ventricular myocytes.¹⁰⁰ In the absence of IBMX, the application of Iso to half of the cell increased I_Ca,L half maximally, corresponding to activation of the channels located in the same part of the cell as the β-AR agonist. When IBMX was added with Iso on half of the cell, the effect of Iso was greatly potentiated because in this condition, LTCCs in the remote part of the cells could be recruited. These results suggest that PDE activity is important for the definition of local cAMP pools involved in the β-AR stimulation of LTCCs. Subsequent studies using ratiometric FRET biosensors to monitor cAMP directly have shown that the second messenger increases preferentially in discrete microdomains corresponding to the dyad region under β-AR stimulation, and that cAMP diffusion is limited by PDE activity.^101,102 Other studies using recombinant cyclic nucleotide gated channels to measure cAMP generated at the plasma membrane identified specific functional coupling of individual PDE families, mainly PDE3 and PDE4, to β₁-AR, β₂-AR, PGE₁-R, and Glu-R as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.⁸⁴

Although PDE4 regulates I_Ca,L in cardiomyocytes,77,84 the molecular identity of the PDE4 regulating the LTCC was unveiled only recently.⁹¹ In mouse cardiomyocytes, PDE4B and PDE4D, but not PDE4A, were found to be part of a Ca_V1.2 signaling complex. However, in mice deficient for the Pde4d gene (Pde4d^–/–), basal or β-AR stimulated I_Ca,L were not different from wild-type mice, whereas in Pde4b^–/– mice the β-AR response of I_Ca,L was increased, together with an increase in cell contraction and Ca²⁺ transients.⁹¹ Upon β-AR stimulation in vivo, catheter-mediated burst pacing triggers ventricular tachycardia in Pde4b^–/– mice but not in wild type.⁹¹ Thus, PDE4B is the main PDE4 isoform regulating cardiac LTCC activity and has a key role during β-AR stimulation of cardiac function. PDE4B, by limiting the amount of Ca²⁺ that enters the cell via the LTCCs, prevents Ca²⁺ overload and arrhythmias.

Role of Phosphatases

Formation of inside-out patches from rabbit ventricular myocytes causes run-down of LTCC activity that is blocked by okadaic acid, a serine/threonine phosphatase inhibitor.¹⁰³ This observation provided early functional evidence that a phosphatase is anchored in close proximity to the channel that counteracts upregulation of Ca_V1.2 by phosphorylation. Later, two major cardiac serine–threonine phosphatases,¹⁰⁴ phosphatase 2A (PP2A) and 2B (PP2B or calcineurin), were found to associate with cardiac Ca_V1.2.^105,106 For PP2A, two attachment sites were identified within the C-terminus of the α1-subunit:^107,108 one region spans residues 1795 to 1818 and the other spans residues 1965 to 1971. PP2B binds immediately downstream of residues 1965 to 1971 without competition between these two phosphatases for binding to this rather narrow region.¹⁰⁸ Injection of a peptide that contains residues 1965 to 1971 and displaces PP2A but not PP2B from endogenous Ca_V1.2 increases basal and β-AR stimulated cardiac I_Ca,L.¹⁰⁸ Similarly, inhibition of PP2B with cyclosporin A or the calcineurin autoinhibitory peptide increases cardiac I_Ca,L.¹⁰⁹ This indicates that anchoring of PP2A and PP2B on cardiac Ca_v1.2 negatively regulates LTCC activity, most likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases.

Ca_V1.2 Involvement in Early and Delayed Afterdepolarizations

According to the Coumel’s triangle, the production of a clinical arrhythmia requires three ingredients: an arrhythmogenic substrate, a trigger factor, and modulation factors of which the most common is the autonomic nervous system.¹¹⁰ In pathologic conditions such as atrial fibrillation (AF), cardiac hypertrophy or heart failure (HF), these factors are united to trigger fatal cardiac arrhythmias. These pathologies are accompanied with structural changes, electrophysiological remodeling, and abnormal β-AR activation at tissue and cellular levels promoting aberrant electrical activities and modulation. Modified excitation-contraction coupling owing to altered calcium homeostasis in pathologic conditions is a major cause of arrhythmias.¹¹¹ As described in the previous paragraph, a fine tuning of the LTCC (i.e., of calcium cycling) and its regulation in discrete subcellular compartments is required to achieve a normal cardiomyocyte function. In physiopathologic conditions, deregulation of this coupling leads to calcium waves and calcium alternans to promote reentrant arrhythmias and triggered activities.^111,112 At the level of the cardiomyocyte in AF¹¹³ or in HF,¹¹⁴ calcium handling is perturbed, leading to afterdepolarizations. When action potential duration (APD) is excessively prolonged, early afterdepolarization (EAD) can occur during the repolarization phase, whereas arrhythmogenic delayed afterdepolarization (DAD) can occur when the cell is fully returned to its resting potential. Non-reentrant mechanisms involve triggered activities from either EADs or DADs. When APD is prolonged, owing to alteration of Na⁺ or K⁺ conductances (as in long QT syndromes or upon antiarrhythmic treatments), I_Ca,L recovery from inactivation occurs during the plateau phase of the AP which causes EADs.¹¹⁵ This is particularly true when I_Ca,L window current is increased upon β-AR activation, thus increasing calcium entry during the plateau phase and allowing its reactivation.^115,116 EADs are also promoted by increased calmodulin-dependent protein kinase II (CaMKII) activity in hypertrophy and HF. CaMKII is activated by increased intracellular calcium levels upon β-AR activation via PKA-dependent and independent mechanisms.^117,118 CaMKII in turn phosphorylates Ca_V1.2 on its Ca_Vβ_2a subunit, triggering afterdepolarizations.^119,120 However, intracellular calcium overload, by activating the reverse mode of the Na⁺/Ca²⁺ exchange, generates a depolarizing current during the late AP phase 2 and phase 3, which is believed to act synergistically with I_Ca,L to induce EADs.^121,122 Similarly, DADs occur at high intracellular calcium load, and their incidence is increased by both rapid pacing and β-AR stimulation.^114,123 The main triggering mechanism for DADs is spontaneous calcium release activating a transient inward current (I_TI) because of excess diastolic calcium handled by sarcolemmal Na⁺/Ca²⁺ exchange. Large enough DADs can eventually sufficiently depolarize the membrane potential to reach threshold and trigger an AP. Again, increased LTCC activity contributes to DAD occurrence and Ca²⁺-evoked arrhythmias. For example, mutations in the LTCC have been associated with a number of inherited arrhythmia syndromes.¹²⁴ Increased LTCC owing to Ca_V1.2 mutations such as those depicted in the Timothy syndrome when reinforced upon β-AR stimulation is a source for intracellular calcium disorders triggering DADs and severe arrhythmias.¹²⁵ These observations demonstrate that a fine tuning of the β-AR modulation of calcium entry via Ca_V1.2 channels in the myocytes is required to maintain proper calcium homeostasis and electrical activity of the heart.

Atrial Fibrillation

Atrial fibrillation (AF) is an extremely common cardiac arrhythmia, most prevalent in elderly people, that is profoundly influenced by the autonomic nervous system, especially by the adrenergic component.¹²⁶ It is accompanied by APD shortening and intracellular calcium homeostasis disorders.^113,127 Decreased depolarizing I_Ca,L contributes to such APD shortening by favoring repolarization thus reentry substrates. If reductions in I_Ca,L have been observed consistently in atrial myocytes from patients with permanent AF^128–130 or from patients in sinus rhythm with a high risk of AF,^131,132 the exact mechanism for such downregulation is not clearly established. This reduction might be primarily the result of transcriptional downregulation of the α_1C-subunit of LTCCs via a Ca²⁺-dependent calmodulin-calcineurin-NFAT system at the cost of the APD reduction observed in AF.¹³³ Besides, it has been also associated with diminished expression of the Ca_Vβ and α₂-δ accessory subunits,¹³⁴ which are essential for α_1C trafficking to the plasma membrane. Normal trafficking of Ca_V1.2 might as well be impaired by a zinc binding protein (ZnT-1) upregulated in patients with AF.¹³⁵ Upregulation of a microRNA, miR-328, has also been correlated to AF producing downexpression of α_1C and Ca_Vβ₁.¹³⁶ Oxidative stress that occurs in AF could also participate to decrease LTCC activity by inducing S-nitrosylation of α_1C¹³⁷ and by promoting its β-AR stimulation to trigger arrhythmogenic EADs.¹³⁸ I_Ca,L amplitude is clearly decreased in AF, but its β-AR regulation is not systematically modified.¹³⁹ Although β-AR receptor density is not impaired in AF¹⁴⁰, polymorphism of β₁-AR occurs in patients with AF, leading to decreased β-AR cascade,¹⁴¹ that could partially explain the decreased LTCC activity in AF. Another plausible mechanism has been proposed as a decreased phosphorylation of the Ca_V1.2 channel because of suppress to the increased phosphatase 1 or phosphatase 2A activities observed in AF.^130,142 This might be also influenced by the hyperphosphorylation by PKA of the peptide inhibitor 1 that is prominent in AF to increase its inhibitory effect on PP-1.^142,143 Furthermore, depressed expression of the major PDE4 isoform detected in human atria, PDE4D, correlates with aging a well-known favoring factor for AF development.⁹⁷ Although PDE3 is the main cAMP-degrading enzyme in human atria, PDE4 substantially contributes to the enzymatic control of β-AR stimulation of I_Ca,L in human atria, and its inhibition leads to arrhythmias because of dysregulated intracellular calcium homeostasis.⁹⁷ All these observations suggest that altered β-AR modulation of Ca_V1.2 channels happens in AF to contribute to such arrhythmia.

Hypertrophy and Heart Failure

In human HF, a chronic activation of β-AR induced by the elevation of circulating catecholamine levels occurs and contributes to the progression of the disease,144,145 stressing the necessity for a strict control of β-AR signaling. This hypothesis is further demonstrated in animal models with exacerbated β-AR/cAMP/PKA signaling and that develop cardiac hypertrophy and heart failure.^146–148 A hallmark of heart failure is β-AR desensitization with a loss of β-AR signaling compartmentation^35,144 and intracellular calcium cycling perturbations,¹¹⁴ accompanied by profound alterations of the structure of the cardiomyocytes notably a loss of the T-tubules.^149,150 Desensitization of β-AR is promoted by its phosphorylation by the G-protein–coupled receptor kinase 2 (GRK2), a serine/threonine kinase upregulated in hypertrophy and HF.¹⁵¹ The number of functional β-AR is reduced in HF, and their localization is changed, with a redistribution to cell crests of the β₂-AR subtype normally localized in the T-tubules, whereas β₁-R remain uniformly distributed at the membrane.⁴⁶ Interestingly, 80% of the Ca_V1.2 channels are located in the T-tubules¹⁵² where they are targeted by the protein BIN-1.¹⁵³ The number of T-tubular LTCCs is decreased in failing myocytes,^154,155 a reduction associated with a down-regulation of BIN-1 in human failing cardiomyocytes.¹⁵⁶ This must contribute to the decreased EC coupling¹⁵⁷ that might be also affected by the architectural reorganization of the dyadic cleft.^157,158 Nonetheless, there is no real consensus in the literature concerning a reduction of I_Ca,L amplitude in failing cardiomyocytes. If the reduction of Ca_V1.2 channels is well described in animals models for HF, it seems largely accepted that I_Ca,L amplitude is maintained especially in failing human cardiomyocytes.^159,160 This apparent discrepancy might be explained by an increased open probability of single Ca_V1.2 channels because of either their increased phosphorylation^161,162 to altered PKA and phosphatase activities^155,163 or increased expression of Ca_Vβ ancillary subunits.^164,165 Furthermore, cardiac remodeling induced by chronic activation of β-AR signaling activates CaMKII,^166,167 which is known to increase the Ca_V1.2 channel activity¹²⁰ and participate in calcium influx remodeling in heart failure.¹⁶⁸ Blunted β-AR stimulation of I_Ca,L in human failing cardiomyocytes has been consistently reported,^162,169 probably because of more abundant heteromeric Gi/oα proteins,^170–172 leading to an increased β₂-R receptor coupling with G_i antagonizing the β₁-AR stimulation of I_Ca,L.¹⁷³ In addition, increased Gi/oα modifies PP1 and PP2A activities, which are two phosphatases that control LTCC phosphorylation.¹⁶³ GRK2 could also contribute to the remodeling of the β-AR stimulation of I_Ca,L in HF, because knockout mice for this kinase appear to be more resistant to adverse remodeling following myocardial infarction, but they demonstrated an increased basal I_Ca,L amplitude and blunted β-AR stimulation.¹⁷⁴ Furthermore, overexpression of βARKct, a peptide derived from the GRK2 C-terminus, is able to increase β-AR stimulation of I_Ca,L independently of PKA in normal and failing cardiomyocytes by sequestering Gβγ proteins.¹⁷⁵ Moreover, expression of a PDE4 isoform—PDE4B, which modulates β-AR stimulation of I_Ca,L⁹¹—has been shown to be decreased in a rat model of compensated hypertrophy,¹⁷⁶ suggesting that not only the production but the enzymatic degradation of the cAMP controlling I_Ca,L is affected in pathological conditions. Thus, in hypertrophy and HF, the β-AR regulation of Ca_V1.2 channels is altered, contributing to a dysregulation of calcium handling promoting calcium-induced arrhythmias. Accurate calcium influx is not only a determinant for normal cardiomyocyte function during the excitation-contraction coupling; recent evidence in the literature highlighted a possible role of Ca_V1.2 channels in gene transcription and prohypertrophic signaling.¹⁷⁷ Few studies have stated that chronic increase calcium influx via Ca_V1.2 channels is deleterious by promoting prohypertrophic signaling pathways. For example, mice overexpressing the α_1C-subunit of Ca_V1.2 channels exhibit cardiac growth and cardiomyopathy hypertrophy¹⁷⁸ and similarly, overexpression of Ca_Vβ_2a leads to cardiac hypertrophy by increasing I_Ca,L, and activation of calcineurin/nuclear factor of activated T cells (NFAT) and CaMKII/HDAC signaling pathways.¹⁷⁹ These assumptions are confirmed by the fact that LTCC blockers can prevent cardiac remodeling in animal subjected to pressure overload¹⁸⁰ and by the attenuation of cardiac hypertrophy observed when the expression of Ca_Vβ₂ subunit is inhibited.¹⁸¹ Astonishingly, decreasing the expression of α_1C in mice also produces cardiac hypertrophy and HF, but this deleterious effect appears not to be connected directly to the diminished I_Ca,L amplitude, but more possibly to the compensatory neuroendocrine stress induced to balance the decreased contractility.¹⁸² Recently, it has been proposed that PKA phosphorylation of the serine 1700 of α_1C relieves its autoinhibition by inducing the dissociation of its proteolytically cleaved and covalently reassociated distal C-terminus from the proximal C-terminus.³⁰ Interestingly, the C-terminus part of α_1C can translocate to the nucleus to act as a transcription factor for its own expression and to activate hypertrophic signaling.¹⁸³ A possible role of its C-terminus in hypertrophy and HF is reaffirmed by recent studies showing that deletion of the terminal part of the α_1C-subunit results in HF in vivo.^32,184 It is tempting to speculate that chronic β-AR stimulation promotes hypertrophy and HF by favoring a permanent dissociation of the C-terminus of the pore-forming subunit of Ca_V1.2 channels to induce cardiac remodeling.

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