KIF5B gene sequence variation and response of cardiac stroke volume to regular exercise

Abstract

A genome-wide linkage scan for endurance training-induced changes in stroke volume detected a quantitative trait locus on chromosome 10p11 in white families of the HERITAGE Family Study. Dense microsatellite mapping narrowed down the linkage region to a 7 Mb area containing 16 known and 14 predicted genes. Association analyses with 90 single nucleotide polymorphisms (SNPs) provided suggestive evidence (P values from 0.03 to 0.06) for association in the kinesin heavy chain (KIF5B) gene locus in the whole cohort. The associations at the KIF5B locus were stronger (P values from 0.001 to 0.008) when the analyses were performed on linkage-informative families only (family-specific logarithm of the odds ratio scores >0.025 at peak linkage location). Resequencing the coding and regulatory regions of KIF5B revealed no new exonic SNPs. However, the putative promoter region was particularly polymorphic, containing eight SNPs with at least 5% minor allele frequency within 1850 bp upstream of the start codon. Functional analyses using promoter haplotype reporter constructs led to the identification of sequence variants that had significant effects on KIF5B promoter activity. Analogous inhibition and overexpression experiments showed that changes in KIF5B expression alter mitochondrial localization and biogenesis in a manner that could affect the ability of the heart to adjust to regular exercise. Our data suggest that KIF5B is a strong candidate gene for the response of stroke volume to regular exercise. Furthermore, training-induced changes in submaximal exercise stroke volume may be due to mitochondrial function and variation in KIF5B expression as determined by functional SNPs in its promoter.

Fig. 1.

Multipoint linkage of stroke volume during steady-state exercise at 50 W (SV50) training response on chromosome 10 in white families of the HERITAGE Family Study. The results are derived from regression-based linkage method implemented in the MERLIN software package using microsatellite panel that included 6 additional markers on the original quantitative trait locus (QTL) region on 10q11.2. LOD, logarithm of the odds ratio.

Fig. 2.

Association between SV50 training response and KIF5B SNP rs211302 (−835) in linkage-positive and linkage-negative subjects.

Fig. 3.

Tissue profile of the human KIF5B by Northern analysis. Two commercial blots (Clontech, Palo Alto, CA) were used: multiple human tissues (MTB) and cardiac (CVD). MTB, multiple tissue Northern blot, Lanes 1–12: brain (whole), heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, leukocyte. CVD, cardiovascular blot, Lanes 1–8: fetal heart, adult heart, aorta, apex of the heart, atrium left, atrium right, ventricle left, ventricle right. The KIF5B transcript is indicated with angled arrows.

Fig. 4.

KIF5B in silico promoter analysis. A: expressed sequence tag assembly shows that KIF5B transcripts all initialize in a discrete locus ∼100 bp upstream of the start of the NCBI reference sequence for the KIF5B mRNA (dotted arrow). The initiation positions are indicated by arrows and the number of transcripts are written above. The position of the single nucleotide polymorphism (SNP) rs1221445 at −444 is highlighted. B: 2000 bp around the KIF5B promoter was analyzed for the presence of CpG islands. CpG dinucleotides are depicted as vertical lines, the identified CpG island is marked with a solid line, and the transcription start site area with an arrow. Numbering of the nucleotides in both panels is in relation to the start codon (i.e., nucleotide preceding ‘A’ of the ATG codon is −1).

Fig. 5.

Functional analyses with KIF5B variants rs211302 (−835) and rs211301 (−809) Transient transfection data of KIF5B haplotype promoter reporter constructs in C2C12 (A) and CHO (B) cells.

Fig. 6.

Functional analyses of KIF5B promoter haplotypes. A: summary of KIF5B promoter haplotypes listing the positions relative to the ATG, the dSNP rs-number, and the frequency. The rare alleles are boxed. B: transient transfection data of KIF5B haplotype promoter reporter constructs and the empty vector in C2C12 cells. Shown are means ± SE of 4 independent experiments with each construct in triplicates (n = 12). Haplotype constructs that share rare alleles are depicted in the same color, haplotype 2 (open bar) contains all common alleles. Post hoc pair-wise comparison P values are written above the bars. ***P < 0.0001 against all other constructs.

Fig. 7.

Effects of kif5b inhibition on mitochondrial content of C2C12 cells. A: immunostaining of cells transfected with negative control scrambled RNA probes (scrRNA), shown in bright green by green fluorescence protein (GFP), had no effect on kif5b expression nor mitochondrial staining. B: cells transfected with antisense siRNA primers against mouse kif5b, shown in bright green by GFP display reduced Kif5b expression and mitochondrial staining. C: siRNA against kif5b led to significant reduction of kif5b mRNA and protein levels (D). E: Ki5fb inhibition with siRNA reduced mitochondrial biogenesis as measured by the decrease of mitochondrial cytochrome b in a dose-dependent manner. *P < 0.05; ***P < 0.001.

Fig. 8.

Effects of kif5b overexpression on mitochondrial content of C2C12 cells. A: immunostaining of cells transfected with the negative control empty vector pcDNA3.1 had no effect on kif5b expression and mitochondrial content. Transfected cells are shown in bright green by GFP. B: Kif5b expression and mitochondrial staining were significantly increased in cells transfected with the kif5b expression construct, identified by the GFP. C: overexpression of kif5b led to significant increase of kif5b mRNA and protein levels (D). E: transient transfection of cells with the kif5b expression construct increased mitochondrial biogenesis as measured by the increase of mitochondrial cytochrome b adjusted by nuclear DNA. *P < 0.05; **P < 0.01.