Localisation of AMPK γ subunits in cardiac and skeletal muscles.

The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

Embryonic expression of AMPK γ subunits and the identification of a novel γ2 transcript variant in adult heart.

AMP-activated protein kinase (AMPK), the key sensor and regulator of cellular energy status, is a heterotrimeric enzyme with multiple isoforms for each subunit (α1/α 2; ß1/ß2; γ1/γ2/γ3). Mutations in PRKAG2, which encodes the γ2 regulatory subunit, cause a cardiomyopathy characterized by hypertrophy and conduction abnormalities. The two reported PRKAG2 transcript variants, γ2-short and γ2-long (encoding 328 and 569 amino acids respectively), are both widely expressed in adult tissues. We show that both γ2 variants are also expressed during cardiogenesis in mouse embryos; expression of the γ3 isoform was also detected unexpectedly at this stage. As neither γ2 transcript is cardiac specific nor differentially expressed during embryogenesis, it is paradoxical that the disease is largely restricted to the heart. However, a recently annotated γ2 transcript, termed γ2-3B as transcription starts at an alternative exon 3b, has been identified; it is spliced in-frame to exon 4 thus generating a protein of 443 residues in mouse with the first 32 residues being unique. It is increasingly expressed in the developing mouse heart and quantitative PCR analysis established that γ2-3B is the major PRKAG2 transcript (~60%) in human heart. Antibody against the novel N-terminal sequence showed that γ2-3B is predominantly expressed in the heart where it is the most abundant γ2 protein. The abundance of γ2-3B and its tissue specificity indicate that γ2-3B may have non-redundant role in the heart and hence mediate the predominantly cardiac phenotype caused by PRKAG2 mutations.

A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis.

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein Mal (also known as TIRAP), encoded by TIRAP (MIM 606252), mediates downstream signaling of TLR2 and TLR4 (refs. 4-6). We report a case-control study of 6,106 individuals from the UK, Vietnam and several African countries with invasive pneumococcal disease, bacteremia, malaria and tuberculosis. We genotyped 33 SNPs, including rs8177374, which encodes a leucine substitution at Ser180 of Mal. We found that heterozygous carriage of this variant associated independently with all four infectious diseases in the different study populations. Combining the study groups, we found substantial support for a protective effect of S180L heterozygosity against these infectious diseases (N = 6,106; overall P = 9.6 x 10(-8)). We found that the Mal S180L variant attenuated TLR2 signal transduction.

Comparison of human and porcine aortic valves.

We compared the anatomy of human and porcine aortic valves. Porcine hearts were collected from the abattoir. Human hearts from patients who had died of non-cardiac causes were examined in the mortuary; only undamaged and anatomically normal hearts were used. Silicon casts were prepared by injecting engineering silicon at 80 mm Hg into the aortic arch. Various features of the aortic valve were measured: circumference, length between the commissural end point and central point of coaptation, surface diameter, and surface area. In total, 12 porcine and 12 human aortic valves were studied. The average circumferences of the human and porcine aortic valves were 8.00 +/- 0.2 (SD) cm and 7.90 +/- 1.0 cm, respectively. The central point of coaptation in human valves was skewed toward the left coronary cusp, whereas in porcine valves it was skewed toward the non-coronary cusp. In human aortic valves, the non-coronary cusp had the largest surface diameter and surface area with mean measurements of 3.6 +/- 0.2 cm and 1.230 +/- 0.228 cm(2), respectively; the left coronary cusp was smallest for the same variables with measurements of 3.1 +/- 0.3 cm and 0.898 +/- 0.357 cm(2). In porcine valves, the right coronary cusp had the largest surface diameter and surface area with mean measurements of 3.9 +/- 0.7 cm and 1.716 +/- 0.81 cm(2), respectively; the non-coronary cusp was the smallest for the same variables with measurements of 2.9 +/- 0.5 cm and 1.023 +/- 0.659 cm(2). These differences suggest that when using porcine valves as transplant material (e.g., stentless valves), geometric considerations, such as commissural length, may be important.