Subunit Expression of the Cardiac L-Type Calcium Channel Is Differentially Regulated in Diastolic Heart Failure of the Cardiac Allograft

Abstract

Background-Left ventricular diastolic dysfunction is a major cause of cardiac allograft failure. Multimeric L-type calcium channels (␣ 1 -, ␣ 2 /␦-, and ␤-subunits) are essential for excitation/contraction coupling in the heart. Their gene expression was studied in allografts that developed diastolic heart failure. Methods and Results-mRNA levels of calcium channel subunits were measured by competitive reverse transcriptasepolymerase chain reaction in microbiopsy samples from the interventricular septum. Size and tissue variabilities between biopsy samples were assessed by determination of cardiac calsequestrin mRNA levels. In the cardiac allografts studied, mRNA levels in microbiopsy samples were considered to represent left ventricular gene expression, because septal and left ventricular gene expression in Northern blots was equivalent, and left ventricles contracted homogeneously. Biopsy samples (nϭ72) were taken from allografts with normal left ventricular end-diastolic pressure (LVEDP; 8 to 13 mm Hg; nϭ30), moderately elevated LVEDP (14 to 18 mm Hg; nϭ26), and elevated LVEDP (19 to 28 mm Hg; nϭ16). Increased LVEDP was related to slowed diastolic relaxation determined by the time constant (r 2 ϭ0.86), whereas systolic performance (dP/dt; ejection fraction) was preserved. With increasing LVEDP, mRNA levels of the pore-forming ␣ 1c -subunit (nϭ15) and of the regulatory ␣ 2 /␦-subunit (nϭ17) remained unchanged but decreased exponentially (r 2 ϭϪ0.83) for the regulatory ␤-subunit (nϭ40). Compared with cardiac allografts with normal LVEDP (nϭ15), ␤-subunit mRNA level was reduced by 75% at elevated LVEDP (nϭ9; Pϭ0.012). In an explanted, diastolically failing cardiac allograft, ␤-subunit expression was reduced correspondingly by 72% and 76% on the mRNA level in septal and left ventricular myocardium and by 80% on the protein level. Conclusions-The downregulated expression of the calcium channel ␤-subunit might contribute to altered calcium handling in diastolically failing cardiac allografts. (Circulation. 1999;100:155-163.)

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

“…The specific probe for the housekeeping gene cardiac calsequestrin hybridized to major transcripts of 2.6 kb in NF and ICM LV mRNA (Fig. 2B) as described before (3,5). Generally, Northern blot intensity of all transcripts detected in RV tissues was less strong compared with LV tissues despite similar total mRNA loading reflecting left ventricular myocyte to matrix dominance.…”

“…Expression data in each specimen were normalized to the respective expression of the housekeeping gene cardiac calsequestrin (3,21), which was also determined by real-time RT-PCR. Real-time RT-PCR for ␤ 2a /␤ 2b was designed with isoform-specific N-terminal sense primers but common C-terminal antisense primer and fluorescent probe.…”

“…[1][2][3][4]. Our studies, however, revealed alterations of auxiliary ␤-subunit expression, which is reduced in diastolic heart failure of cardiac allografts (3) and possibly in end stage heart failure (5).…”

“…[1][2][3][4]. Our studies, however, revealed alterations of auxiliary ␤-subunit expression, which is reduced in diastolic heart failure of cardiac allografts (3) and possibly in end stage heart failure (5). Down-regulation of ␤-subunit mRNA expression is a phenomenon not restricted to cardiac tissue, because similar observations were made in pancreatic islets of diabetic rats (6).…”

“…In human myocardium expression of three different ␤-subunits genes (␤ 1 -␤ 3 ) (8 -10) was demonstrated at the mRNA and the protein level (3,10). In human non-failing (NF) 1 left ventricular (LV) mRNA our RT-PCR experiments with degenerated primers complementary to coding sequences with high similarity between ␤ 2 -and ␤ 3 -genes generated three amplification products of 650, 503, and 483 bp.…”