Calcineurin and Calcium/Calmodulin-dependent Protein Kinase Activate Distinct Metabolic Gene Regulatory Programs in Cardiac Muscle

Abstract

To learn more about the targets of Cn (Cn) and calcium/calmodulin-dependent protein kinase in cardiac muscle, we investigated their actions in cultured cardiac myocytes and the hearts of mice in vivo. Adenoviral-mediated expression of constitutively active forms of either pathway induced expression of peroxisome proliferator-activated receptor ␥ coactivator 1␣, a transcriptional coactivator involved in the control of multiple cellular energy metabolic pathways in cardiac myocytes. Transcriptional profiling studies demonstrated that Cn and calcium/calmodulin-dependent protein kinase activate distinct but overlapping metabolic gene regulatory programs. Expression of the nuclear receptor, peroxisome proliferator-activated receptor ␣, was markedly increased by Cn, but not calcium/calmodulin-dependent protein kinase, providing one mechanism whereby cellular fatty acid utilization genes are selectively activated by Cn. Transfection experiments demonstrated that Cn directly activates the mouse peroxisome proliferator-activated receptor ␣ gene promoter. Co-transfection "add-back" experiments demonstrated that the transcription factors, myocyte enhancer factors 2C or 2D, were sufficient to confer Cn-mediated activation of the peroxisome proliferatoractivated receptor ␣ gene. Cn was also shown to directly activate a known peroxisome proliferator-activated receptor ␣ target, muscle-type carnitine palmitoyltransferase I, providing a second mechanism by which Cn activates genes of cellular fatty acid utilization. Lastly, the gene expression of peroxisome proliferator-activated receptor ␥ coactivator 1␣ and peroxisome proliferator-activated receptor ␣ was reduced in the hearts of mice with cardiac-specific ablation of the Cn regulatory subunit. These data support a role for calcium-triggered signaling pathways in the regulation of cardiac energetics and identify pathway-specific control of metabolic targets.Skeletal muscle fiber types are defined by contractile protein isoform composition and energy metabolic properties. Slowtwitch muscle fibers possess greater mitochondrial volume supporting higher oxidative metabolic capacity compared with fast-twitch fibers. Muscle metabolic phenotype is plastic, responding to numerous external stimuli. Calcium signaling serves an important role in the adaptive response of skeletal muscle to external stimuli, including fiber type determination. Evidence has emerged that calcium-triggered regulatory pathways acting through Cn (Cn), 1 a serine/threonine protein phosphatase, and calcium/calmodulin-dependent protein kinases (CaMK), serve a major role in determining the functional and metabolic phenotype of skeletal muscle by transducing alterations in cytosolic calcium concentration. In support of this, Chin et al. (1) demonstrated that a constitutively active form of Cn (Cn*) is capable of activating transcription of slow fiberspecific gene promoters. Similarly, overexpression of Cn* in skeletal muscle results in an increase in slow muscle fiber types (2), and in CnA␣ and A␤ null mice there ...

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