A. Targeted disruption of the voltage-dependent calcium channel ␣2/␦-1-subunit. Cardiac L-type voltage-dependent Ca 2ϩ channels are heteromultimeric polypeptide complexes of ␣1-, ␣2/␦-, and ␤-subunits. The ␣2/␦-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in ␣2/␦-subunits of voltage-dependent Ca 2ϩ channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the ␣2/␦-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of ␣2/␦-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca 2ϩ current peak current amplitude. This is a novel model for studying the function of the ␣2/␦-1-subunit and will be of importance in the development of new pharmacological therapies. cardiac calcium channel; murine knockout model; gabapentin binding; myocardial contractility CARDIAC L-type voltage-dependent Ca 2ϩ channels (L-VDCCs) are heteromultimeric polypeptide complexes of ␣ 1 -, ␣ 2 /␦-, and ␤-subunits. The ␣ 1 -subunit is autoregulatory and harbors the channel pore, gating machinery, and modulatory drug binding sites (30). The accessory subunits (␣ 2 /␦ and ␤) affect channel kinetics and are involved in the trafficking and insertion of the ␣ 1 -subunit into the membrane. The ␣ 2 -subunit is closely associated with an extracellular loop of the ␣ 1 -subunit (15) and linked to a small protein called ␦ (2, 9). Both the ␣ 2 and ␦ are encoded by the same gene, separated by proteolytic cleavage, and extracellularly linked through a disulfide bridge (9). Currently, four ␣ 2 /␦-subunits, each encoded by separate genes, have been identified (4). The ␣ 2 /␦-1, originally cloned from skeletal muscle (10), is ubiquitously distributed (18), with high levels of protein expression in brain, heart, skeletal, and

Postconditioning (POC), a novel strategy of cardioprotection against ischemia-reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that POC requires the activation of both JAK-STAT and RISK signaling. Langendorff-perfused mouse hearts were subjected to 30 min of global ischemia and 40 min of reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG 490, a JAK2 inhibitor, Stattic, a specific STAT3 inhibitor, or LY-294002, a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/dt(max), mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated (p-) STAT3 and Akt expression was analyzed by Western immunoblotting. POC enhanced myocardial functional recovery and increased expression of p-STAT3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT3 inhibition decreased expression of both p-STAT3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT3 phosphorylation. Interestingly, STAT3 KO hearts undergoing POC exhibited improved ischemic tolerance compared with KO non-POC hearts. POC induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective POC without PI3K-Akt activation.

Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function. We previously developed two transgenic mouse models that carry FHC-associated mutations in alpha-tropomyosin (TM): FHC alpha-TM175 mice show patchy areas of mild ventricular disorganization and limited hypertrophy, whereas FHC alpha-TM180 mice exhibit severe hypertrophy and fibrosis and die within 6 mo. To obtain a better understanding of the molecular mechanisms associated with the early onset of cardiac hypertrophy, we conducted a detailed comparative analysis of gene expression in 2.5-mo-old control, FHC alpha-TM175, and alpha-TM180 ventricular tissue. Results show that 754 genes (from a total of 22,600) were differentially expressed between the nontransgenic (NTG) and the FHC hearts. There are 178 differentially regulated genes between NTG and the FHC alpha-TM175 hearts, 388 genes are differentially expressed between NTG and FHC alpha-TM180 hearts, and 266 genes are differentially expressed between FHC alpha-TM175 and FHC alpha-TM180 hearts. Genes that exhibit the largest increase in expression belong to the "secreted/extracellular matrix" category, and those with the most significant decrease in expression are associated with "metabolic enzymes." Confirmation of the microarray analysis was conducted by quantitative real-time PCR on gene transcripts commonly associated with cardiac hypertrophy.

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Despite previous reports of improved cardiac function in heart failure models after SERCA intervention, our results advocate the need to elucidate the involvement of augmented Ca(2+) cycling in arrhythmogenesis.