Transcriptional Control of Muscle Cell Excitation-Contraction Coupling: The role of activity and mitochondrial function

Heart disease remains a leading cause of death worldwide, and key to finding new treatments and improving prognosis of patients is understanding cardiac cell function at the cellular level. When we understand how normal cell processes work and can identify where things go wrong in disease, we can try to isolate the problems and address them with specially targeted drugs to improve cardiac function and slow down or prevent the development of cardiac disease. While it may seem a long way from the laboratory to the patient, basic research on cardiac cells is crucial for medical science to move forward.

Muscle cells require large amounts of energy to power contractions and maintain ion gradients, and this energy is provided by the powerhouses of the cell – mitochondria. When mitochondrial function is impaired, muscle performance suffers. Mitochondrial dysfunction has been associated with decreased cardiac and skeletal muscle performance during aging and the pathogenesis of diseases, such as diabetic cardiomyopathy, hypertrophy, ischemia/reperfusion injury, and heart failure.

In this thesis, the function of muscle cells was studied at the level of gene expression under different stress conditions. Two models of mitochondrial dysfunction were used: a mouse model of mitochondrial myopathy, in which the important transcription factor Tfam was knocked out, and cultured cardiomyocytes where mitochondrial function was impaired using the chemical FCCP. Interestingly, in both Tfam muscle cells and cultured cardiac cells, calsequestrin was downregulated at the gene and protein levels. Calsequestrin is the main calcium buffering protein within the SR, from where contraction-initiating calcium oscillations originate. Its expression is strictly regulated due to its importance in maintaining SR calcium load and its regulatory effects on SR calcium release via the ryanodine receptors. However, mitochondrial stress caused significant downregulation of calsequestrin levels, which in turn led to decreased SR calcium stores and reduced force production in muscle cells, and impaired calcium handing and contraction in cultured cardiac cells. As muscle contraction consumes large amounts of energy, it makes sense that cells may attempt to limit calcium release in times of mitochondrial stress in order to prolong survival.

In cardiac disease, a common underlying aspect is increased intracellular calcium levels between contractions. This causes an ionic imbalance that can lead to impaired excitation-contraction coupling (ECC), decreased contractile ability, hypertrophic remodeling, and arrhythmias. This thesis also studied the effects of increased intracellular calcium levels on cardiomyocyte transcriptional regulation, in order to identify feedback mechanisms that may prevent intracellular calcium surplus. In cultured cardiac cells, calcium-calmodulin kinase (CaMKII), which is activated by increased intracellular calcium levels, decreased Cacna1c expression. Cacna1c codes for the pore-forming subunit of the L-type calcium channel (LTCC). Upon activation of LTCC, a small amount of calcium enters the cell, triggering release of intracellular SR calcium stores and leading to contraction of the cell. Cacna1c transcription was downregulated via binding of the transcriptional repressor DREAM to a putative downstream regulatory element (DRE) in the Cacna1c promoter. By altering LTCC levels, the cell can adjust the amount of calcium entry through LTCCs in response to the calcium levels sensed by CaMKII. This calcium-CaMKII-DREAM-LTCC cascade may be an important feedback mechanism whereby cardiac cells regulate intracellular calcium levels to prevent calcium overload.