Cardiac L-type Calcium Channel β-Subunits Expressed in Human Heart Have Differential Effects on Single Channel Characteristics

l-Type calcium channels are multiprotein complexes composed of pore-forming (CaV1.2) and modulatory auxiliary α2δ- and β-subunits. We demonstrate expression of two different isoforms for the β2-subunit (β2a, β2b) and the β3-subunit (β3a, β3trunc) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of β2b transcripts prevails in the order of > β3 >> β2a. The expressed cardiac full-length β3-subunit is identical to the β3a-isoform, and β3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium β3trunc expression increases whereas overall β3 expression remains unchanged. Heterologous coexpression studies demonstrated that β2 induced larger currents through rabbit and human cardiac CaV1.2 pore subunits than β3 isoforms. All β-subunits increased channel availability at single channel level, but β2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac β-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various β-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure. l-Type calcium channels are multiprotein complexes composed of pore-forming (CaV1.2) and modulatory auxiliary α2δ- and β-subunits. We demonstrate expression of two different isoforms for the β2-subunit (β2a, β2b) and the β3-subunit (β3a, β3trunc) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of β2b transcripts prevails in the order of > β3 >> β2a. The expressed cardiac full-length β3-subunit is identical to the β3a-isoform, and β3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium β3trunc expression increases whereas overall β3 expression remains unchanged. Heterologous coexpression studies demonstrated that β2 induced larger currents through rabbit and human cardiac CaV1.2 pore subunits than β3 isoforms. All β-subunits increased channel availability at single channel level, but β2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac β-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various β-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure. l-Type calcium channels are key elements of cardiac excitation-contraction coupling. In the human heart calcium channels consist of the ion conducting pore (α1C-subunit or Cav1.2) and two auxiliary subunits (β, α2δ), which modulate electrophysiological and pharmacological properties. The Cav1.2-subunit also harbors the binding sites for the calcium modulatory drugs. In distinct cardiac diseases such as severe chronic atrial fibrillation or hypertrophic obstructive cardiomyopathy Cav1.2-subunit expression is altered, but in human diastolic or end stage heart failure most studies have demonstrated no change (see Refs. 1Hersel J. Jung S. Mohacsi P. Hullin R. Basic Res. Cardiol. 2002; 97: I/4Google Scholar, 2Richard S. Leclercq F. Lemaire S. Piot C. Nargeot J. Cardiovasc. Res. 1998; 37: 300-311Crossref PubMed Scopus (116) Google Scholar, 3Hullin R. Asmus F. Ludwig A. Hersel J. Boekstegers P. Circulation. 1999; 100: 155-163Crossref PubMed Scopus (46) Google Scholar, 4Wagner J.A. Reynolds I.J. Weisman H.F. Dudeck P. Weisfeldt M.L. Snyder S.H. Science. 1986; 232: 515-518Crossref PubMed Scopus (112) Google Scholar). Our studies, however, revealed alterations of auxiliary β-subunit expression, which is reduced in diastolic heart failure of cardiac allografts (3Hullin R. Asmus F. Ludwig A. Hersel J. Boekstegers P. Circulation. 1999; 100: 155-163Crossref PubMed Scopus (46) Google Scholar) and possibly in end stage heart failure (5Schröder F. Handrock R. Beuckelmann D.J. Hirt S. Hullin R. Priebe L. Schwinger R.H.G. Weil J. Herzig S. Circulation. 1998; 98: 969-976Crossref PubMed Scopus (233) Google Scholar). Down-regulation of β-subunit mRNA expression is a phenomenon not restricted to cardiac tissue, because similar observations were made in pancreatic islets of diabetic rats (6Iwashima Y. Abiko A. Ushikubi F. Hata A. Kaku K. Sano H. Eto M. Biochem. Biophys. Res. Commun. 2001; 280: 923-932Crossref PubMed Scopus (26) Google Scholar). Although an altered stoichiometry of β-subunits relative to the Cav1.2-subunits theoretically suggests variation in calcium inward current, we and others have detected no change in whole-cell calcium inward currents in cardiomyocytes isolated from human failing heart. Instead, calcium channel inward current was even paradoxically increased at the single channel level and could not be further augmented by cAMP-dependent phosphorylation (5Schröder F. Handrock R. Beuckelmann D.J. Hirt S. Hullin R. Priebe L. Schwinger R.H.G. Weil J. Herzig S. Circulation. 1998; 98: 969-976Crossref PubMed Scopus (233) Google Scholar). Because products from all four β-subunit genes modulate the ion conducting pore in a specific manner (7Hofmann F. Lacinova L. Klugbauer N. Rev. Physiol. Biochem. Pharmacol. 1999; 139: 33-87Crossref PubMed Google Scholar), we decided to study cardiac β-subunit gene expression in the human heart as a plausible reason for increased single calcium channel activity in heart failure. In human myocardium expression of three different β-subunits genes (β1-β3) (8Freise D. Himmerkus N. Schroth G. Trost C. Weissgerber P. Freichel M. Flockerzi V. Biol. Chem. 1999; 380: 897-902Crossref PubMed Scopus (15) Google Scholar, 9Collin T. Wang J.J. Nargeot J. Schwartz A. Circ. Res. 1993; 72: 1337-1344Crossref PubMed Scopus (56) Google Scholar, 10Haase H. Kresse A. Hohaus A. Schulte H.D. Maier M. Osterziel K.J. Lange P.E. Morano I. J. Mol. Med. 1996; 74: 99-104Crossref PubMed Google Scholar) was demonstrated at the mRNA and the protein level (3Hullin R. Asmus F. Ludwig A. Hersel J. Boekstegers P. Circulation. 1999; 100: 155-163Crossref PubMed Scopus (46) Google Scholar, 10Haase H. Kresse A. Hohaus A. Schulte H.D. Maier M. Osterziel K.J. Lange P.E. Morano I. J. Mol. Med. 1996; 74: 99-104Crossref PubMed Google Scholar). In human non-failing (NF) 1The abbreviations used are: NF, non-failing; LV, left ventricular; RT, reverse transcriptase; ICM, ischemic cardiomyopathy; RA, right atrium; RV, right ventricle; CHO, Chinese hamster ovary; HEK, human embryonic kidney; GFP, green fluorescent protein; ANOVA, analysis of variance; nn, nucleotide. left ventricular (LV) mRNA our RT-PCR experiments with degenerated primers complementary to coding sequences with high similarity between β2- and β3-genes generated three amplification products of 650, 503, and 483 bp. The 650- and 503-bp amplification products are sequence-identical to β2 and β3 coding sequences (β2: GenBank™ accession number AF423189, nn 97–746; β3: GenBank™ accession number X76555, nn 135–637). The 483-bp amplification product contained a deletion of 20 nucleotides matching exon 6 of the β3-gene (11Yamada Y. Masuda K. Li Q. Ihara Y. Kubota A. Miura T. Nakamura K. Fujii Y. Seino S. Seino Y. Genomics. 1995; 27: 312-319Crossref PubMed Scopus (37) Google Scholar). At least two different isoforms of the human β2-subunit gene are expressed in the human heart (β2a, β2b) (12Yamaguchi H. Okuda M. Mikala G. Fukasawa K. Varadi G. Biochem. Biophys. Res. Commun. 2000; 267: 156-163Crossref PubMed Scopus (25) Google Scholar, 13Colecraft H.M. Alseikhan B. Takahashi S.X. Chaudhuri D. Mittman S. Yegnasubramanian V. Alvania R.S. Johns D.C. Marban E. Yue D.T. J. Physiol. 2002; 541: 435-453Crossref PubMed Scopus (187) Google Scholar) that differ with respect to their short amino termini. Our Northern blot experiments revealed transcripts for β2a-, β2b-, and β3-subunits in human heart tissue; therefore, we assessed quantitative expression of these β-subunits by real-time PCR, which demonstrated substantially different expression levels with β2b > β3 » β2a. To investigate the physiological effects of these β-subunits, we first cloned both the full-length coding sequence of the β3-subunit (β3a) and the isoform containing the deletion of exon 6 (20 nn) (β3trunc) from human heart. Deletion of exon 6 results in truncation of the protein, and, interestingly, βtrunc is up-regulated in heart failure. Because of the high homology of the rabbit β2a-subunit to the human β2b we used this subunit together with the two cloned human β3-subunit isoforms to study the functional impact of these β-subunits onto the calcium inward current at the single channel level. Electrophysiological studies were designed to answer the following questions. 1) Do human cardiac β3-subunits functionally coexpress with pore subunits from rabbit heart (Cav1.2a), rabbit smooth muscle (Cav1.2b), and human heart (Cav1.2)? Is there a difference in quality and extent of their modulation when compared with a β2-subunit? 2) Does alternative splicing of the β3-subunits in heart failure explain the increase in single channel activity? These questions were addressed by transient coexpression in cell lines stably expressing CaV1.2 isoforms. Tissue Specimens—After local ethics committee approval, human myocardium specimens were obtained from explanted NF hearts not transplanted for technical reasons or from patients receiving orthotopic heart transplantation because of end-stage heart failure due to ischemic cardiomyopathy (ICM). All specimens were frozen directly after explantation. PCR-based Analysis of β-Subunit Gene Expression in Human Myocardium—The experiment is not depicted in this primers complementary to coding sequences of high similarity between human β2- and β3-subunits were obtained from RT-PCR at and was with of mRNA isolated from NF human myocardium in containing primers and of products were and 483-bp were by and of expression in human heart specimens was by RT-PCR at and and GenBank™ accession number with of mRNA isolated from and products were (see and analysis of was by were with the real-time of in (β2a, or of the and β2b expression was with and fluorescent GenBank™ accession number fluorescent GenBank™ accession number and primers GenBank™ accession number GenBank™ accession number complementary to the short termini. was with and fluorescent and GenBank™ accession number for and β3 were for at and in for and β3 were and were by expression was and and GenBank™ accession number in at at and with of of amplification were by and for cardiac by Northern the β3-subunit of mRNA isolated with and from human right right ventricle and obtained from hearts were used for Northern blot mRNA and Northern blot experiments were as R. D. Freichel M. M. N. F. Flockerzi V. J. PubMed Scopus Google Scholar). Northern were at high at in with of a from the 503-bp amplification product Northern were for at in in and at in ICM, was at for Northern the β2-subunit of NF mRNA were and with and β2b and GenBank™ accession number and GenBank™ accession number were and were and were generated by were as was was All were at and at in with and were at in were and at cardiac blot was and as for but with a specific for human cardiac GenBank™ accession number was with for at at and at was 6 at of sequence was cloned two of primers from and and and and (β3a) or a at the position to a The full-length was the and the generated and is sequence-identical to and and transient were as J.A. A. N. N. H. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, R. R. Klugbauer N. F. Herzig S. J. Physiol. 1999; Scopus Google Scholar). In Chinese hamster were stably with the cloned from rabbit heart A. E. Flockerzi V. F. R.S. Circ. Res. 1993; PubMed Scopus Google Scholar) or with the from rabbit E. R. T. J. A. F. Flockerzi V. J. PubMed Scopus (46) Google Scholar). Human embryonic were stably with the human cardiac Cav1.2-subunit D. Mikala G. A. B. M. Wang J.J. D. Varadi G. Schwartz A. 1993; PubMed Scopus Google Scholar). experiments with pore subunits were in at a between and and used within after expression cloned human and rabbit and sequences were or were with the the different β-subunits together with rabbit muscle R. E. A. Schwartz A. M. Science. PubMed Scopus Google and green protein was by with and the at a of J.A. A. N. N. H. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). were in with and and both Electrophysiological were after Electrophysiological and single channel currents through channels were at experiments were in containing were with currents were at by from a of to various as were at and at and currents were by a currents were the of a was as the current at the end of a relative to peak The was used for and channel and analysis were as (5Schröder F. Handrock R. Beuckelmann D.J. Hirt S. Hullin R. Priebe L. Schwinger R.H.G. Weil J. Herzig S. Circulation. 1998; 98: 969-976Crossref PubMed Scopus (233) Google Scholar). were with containing with were with with calcium channels were in the of at and or were used for and were the channel activity for at least in most and currents were and were by the The availability of containing at least channel the as the relative of the and the peak current obtained were from single channel and In the were for the number of channels in the was as the current by the current was by through The availability was by the is the of The was the of the number of by number of and were in of and closed were obtained by and of the β-subunits single channel were by the of a pore subunit and with and or a β-subunit were by and for was All are as β2- and to distinct transcripts in Northern with human NF and The to two transcripts in RA, RV, and in both human NF and The of the transcripts with respect to and in NF RA, in NF RV, in NF LV, in RA, in RV, and in whereas the was in all heart but in the Because no alternative splicing within the coding sequence was with of β3 by be because of sequence differences in the or transcripts detected by the were of and in mRNA whereas the to transcripts in the Because and β2b coding sequences differ with respect to their short contained of we the of the transcripts detected a coding sequence to human and marked transcripts of similar and also in NF that is in The specific for the gene cardiac to transcripts of in NF and mRNA as (3Hullin R. Asmus F. Ludwig A. Hersel J. Boekstegers P. Circulation. 1999; 100: 155-163Crossref PubMed Scopus (46) Google Scholar, F. Handrock R. Beuckelmann D.J. Hirt S. Hullin R. Priebe L. Schwinger R.H.G. Weil J. Herzig S. Circulation. 1998; 98: 969-976Crossref PubMed Scopus (233) Google Scholar). Northern blot of all transcripts detected in was compared with similar mRNA left ventricular to of the β2a-, β2b-, and β3-subunit mRNA expression was by real-time RT-PCR in NF mRNA and mRNA of Expression in were to the expression of the gene cardiac (3Hullin R. Asmus F. Ludwig A. Hersel J. Boekstegers P. Circulation. 1999; 100: 155-163Crossref PubMed Scopus (46) Google Scholar, G. Cardiovasc. Res. 1998; 37: PubMed Google Scholar), which was also by real-time RT-PCR for was designed with primers but and fluorescent Because we detected no differences of and β3 when NF and LV, were In the specimens the β2b number was which is the number of RT-PCR for the β3 detected in the specimens which was different from or β2b of and cardiac cardiac were to and were (see a for mRNA number was the of a of reverse M. B. B. M. P. P. D. J. Biol. Chem. 267: PubMed Google Scholar). of the products demonstrated amplification product of The full-length cloned from human NF mRNA by RT-PCR of and amino and this β3 is identical to the isoform cloned from human accession number for exon 6 deletion (20 which results in a stop of translation at nucleotide position no splicing product of the β3-gene was in human heart. similar truncation was also in M. Flockerzi V. J. Biochem. 1996; PubMed Scopus Google Scholar). Because a in as demonstrated for H. D. Marban E. Science. 2000; PubMed Scopus Google Scholar), we further the of β3trunc by RT-PCR experiments primers in mRNA isolated from NF and In the specimens in the of isoform expression from in NF to in stably expressing the different of the calcium channel pore were with together with rabbit or the cloned human or The rabbit was for our coexpression experiments because of high homology to the which in the human heart is expressed when compared with human β2a. increased current the and increased the of at the of peak current for and as in as (12Yamaguchi H. Okuda M. Mikala G. Fukasawa K. Varadi G. Biochem. Biophys. Res. Commun. 2000; 267: 156-163Crossref PubMed Scopus (25) Google Scholar, H. M. M. Fukasawa K. Schwartz A. Varadi G. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). Although the effects with of rabbit and human β3-subunits were the effects of and of β3trunc in were and of effects of was the single channel from single channel experiments the pore-forming subunit are depicted in channel activity was in from as from with and and β3trunc increased the of within a within such In rabbit to marked increases in both and to a higher increase in the peak current of the These were Analysis of gating in revealed that the closed is the most by rabbit whereas human or β3trunc have no closed analysis that the in closed is because of a of the of the revealed an increase in the of the but in the of rabbit channel of calcium channels from stably expressing rabbit cardiac in a To the marked modulation by rabbit is restricted to the cardiac pore we the of and the smooth muscle in and similar results were found as with both β-subunits increased availability currents were by because of a rapid gating and to was which explain this these a of the β-subunit pore properties. with and with and a of activity of similar to our observations M. Handrock R. A. F. Herzig S. Pharmacol. 1999; PubMed Scopus Google channel of calcium channels from stably expressing rabbit smooth muscle in a To account for the functional differences found the of human A. H. J. Biol. Chem. PubMed Scopus Google Scholar, Y. K. V. P. J. Mol. Cardiol. 2000; PubMed Scopus Google Scholar), we the pore-forming subunit expressed in human myocardium D. Mikala G. A. B. M. Wang J.J. D. Varadi G. Schwartz A. 1993; PubMed Scopus Google Scholar). We expressed in for these studies, which to a because of The of pore as as their modulation by rabbit and human β3 isoforms were with of the with the cell experiments was but not the β3-subunits increased and to be by because of a change of the first was by β3trunc no effects and a position these The of β3trunc availability and peak current short of the of in this expression we a of to of gating the of the modulation of the pore subunit by and β3trunc the of was to the was not as with and even with could not be because of R. F. Hirt S. A. C. Herzig S. Cardiovasc. Res. 1998; 37: PubMed Google Scholar). that the of is larger with β2a. modulation by and were similar channel of calcium channel complexes from stably expressing human cardiac in a In the present we expression of two β2-subunit isoforms (β2a, β2b) and of two β3-subunits (β3a, β3trunc) in the human heart. The detected distinct transcripts of and in left ventricular mRNA from human non-failing and failing ischemic cardiomyopathy whereas the to a of in both Because of the short of the coding contained sequences to that might to To the of the we used a complementary to the coding sequence to both and detected transcripts of similar of the is Our detected different from the by (8Freise D. Himmerkus N. Schroth G. Trost C. Weissgerber P. Freichel M. Flockerzi V. Biol. Chem. 1999; 380: 897-902Crossref PubMed Scopus (15) Google Scholar) demonstrated in human heart mRNA a of and a of when a β2 in the detected from or of the various in our Northern the of transcripts of similar by two different that sequences of the mRNA was Our marked full-length coding sequences of the and β2b are and We of our mRNA because and β2b real-time RT-PCR from the mRNA in single amplification products amplification of and coding these transcripts of the mRNA the of the β2 gene In to studies that not β3-subunit mRNA expression in human mRNA T. P. S. C. P. P. Nargeot J. J. Biochem. PubMed Scopus (46) Google Scholar), we detected transcripts of and in RA, RV, and from both human non-failing and failing ischemic myocardium. of were also for human T. P. S. C. P. P. Nargeot J. J. Biochem. 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PubMed Google in human heart expression of the gene of be to the in the of the and β3-subunit isoforms be from our gene expression studies, and experiments might also be by their of this we addressed this at the functional level by expressing the and in that stably the and human The calcium currents obtained by coexpression with the β3 isoforms were compared with currents induced by rabbit because of homology to the human the and to a extent the β3trunc functional impact when with the cardiac pore-forming the extent of modulation by was substantially than with β2a. difference is not because of a between the rabbit and human because similar were obtained with both rabbit and human cardiac pore-forming We also to effects of a different of the subunit all technical expression of were and, the of single channel modulation by and to this Although and increased channel a and stimulation of the gating properties. of the of as with an increased gene have both in that our single channel analysis could be by of a of channels in the even in no were this even the between and because we with in the of β2a. The that the of the of channel is in a experiment with but in of R. Biophys. J. PubMed Scopus Google Scholar). The electrophysiological between β-subunits is not were in a increase of whole-cell currents than β3-subunits H.M. R. Yue D.T. Circ. Res. 2000; PubMed Scopus Google Scholar). A. I. Biophys. J. 1998; Scholar) and a H.M. Alseikhan B. Takahashi S.X. Chaudhuri D. Mittman S. Yegnasubramanian V. Alvania R.S. Johns D.C. Marban E. Yue D.T. J. Physiol. 2002; 541: 435-453Crossref PubMed Scopus (187) Google Scholar) that β-subunit isoforms be closed at the single channel level, a in with our The functional of splicing of β3-subunits explain the increase in channel activity as in human heart failure (5Schröder F. Handrock R. Beuckelmann D.J. Hirt S. Hullin R. Priebe L. Schwinger R.H.G. Weil J. Herzig S. 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Res. 2002; PubMed Scopus Google Scholar). In β-subunit gene products modulate cardiac calcium channel in an and is to not the of mRNA or protein of these subunits but the relative of the studies such β-subunit These could as a of altered single channel behavior in heart which in excitation-contraction coupling. We and for technical for with the of the and F. and N. Klugbauer of for cell lines and