A.Ludwig and X.Zong contributed equally to this work Cardiac pacemaking is produced by the slow diastolic depolarization phase of the action potential. The hyperpolarization-activated cation current (I f ) forms an important part of the pacemaker depolarization and consists of two kinetic components (fast and slow). Recently, three full-length cDNAs encoding hyperpolarization-activated and cyclic nucleotide-gated cation channels (HCN1-3) have been cloned from mouse brain. To elucidate the molecular identity of cardiac pacemaker channels, we screened a human heart cDNA library using a highly conserved neuronal HCN channel segment and identified two cDNAs encoding HCN channels. The hHCN2 cDNA codes for a protein of 889 amino acids. The HCN2 gene is localized on human chromosome 19p13.3 and contains eight exons spanning~27 kb. The second cDNA, designated hHCN4, codes for a protein of 1203 amino acids. Northern blot and PCR analyses showed that both hHCN2 and hHCN4 are expressed in heart ventricle and atrium. When expressed in HEK 293 cells, either cDNA gives rise to hyperpolarization-activated cation currents with the hallmark features of native I f . hHCN2 and hHCN4 currents differ profoundly from each other in their activation kinetics, being fast and slow, respectively. We thus conclude that hHCN2 and hHCN4 may underlie the fast and slow component of cardiac I f , respectively.
Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L‐type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.
Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.
Background-Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1␣ (also called ␣-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. Methods and Results-In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1. By developing a TPM1-specific antibody, we found that the TPM1 protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1 in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1. Incorporation of increased levels of TPM1 protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1 compared with TPM1␣. Conclusions-This functional analysis of TPM1 provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients. (Circulation. 2010;121:410-418.)Key Words: cardiomyopathy Ⅲ contractility Ⅲ heart failure Ⅲ myocardial contraction T he heart adapts to different challenges presented by an array of mechanical, hormonal, and nutritional signals in the process of maintaining its circulatory function. Isoform switching of sarcomeric proteins is 1 mode the heart uses to adapt to those challenges, along with alterations in the relative abundance and phosphorylation status of contractile and regulatory proteins. 1 These changes in isoform expression and phosphorylation status also play an essential role during cardiac development and in response to cardiac hypertrophy and heart failure (HF). Although sarcomeric protein isoforms are subject to developmental regulation, cardiomyopathy and HF primarily elicit changes in thick filament protein isoforms. 2 The only thin filament protein to change isoform expression in the failing human heart is troponin T. 3,4 Furthermore, altered phosphorylation of troponin I, myosin binding protein C, and other sarcomeric proteins has dramatic effects on cardiac function in the failing human myocardium. 5 Editorial see p 351 Clinical Perspective on p 418To understand the specific role of another thin filament protein, tropomyosin (TM), in the normal and the pathological heart, it is essential to define the TM isoform expression profile. Tropomyosins comprise a family of actin-binding proteins encoded by 4 different genes (TPM1, TPM2, TPM3, and TPM4). Each gene uses alternative splicing, alternative promoters, and differential processing to encode multiple striated muscle, smooth muscle, and cytoskeletal transcripts. For example, the TPM1...
The complete amino acid sequence of the receptor for organic calcium channel blockers (CaCB) from rabbit lung has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CaCB-sensitive high voltage activated calcium channel in Xenopus oocytes.
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