Inhibition of mitochondrial permeability transition by deletion of the ANT family and CypD

The mitochondrial permeability transition pore (MPTP) has resisted molecular identification. The original model of the MPTP that proposed the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component was challenged when mitochondria from Ant1/2 double null mouse liver still had MPTP activity. Because mice express three Ant genes, we reinvestigated whether the ANTs comprise the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPTP formation, and when also given cyclosporine A (CsA), the MPTP was completely inhibited. Moreover, liver mitochondria from mice with quadruple deletion of Ant1, Ant2, Ant4, and Ppif (cyclophilin D, target of CsA) lacked Ca2+-induced MPTP formation. Inner-membrane patch clamping in mitochondria from Ant1, Ant2, and Ant4 triple null mouse embryonic fibroblasts showed a loss of MPTP activity. Our findings suggest a model for the MPTP consisting of two distinct molecular components: The ANTs and an unknown species requiring CypD

The effects of RYR2 gene deletion on cardiac function and metabolism

The cardiac ryanodine receptor 2 (RYR2) is a sarcoplasmic reticulum Ca²⁺ release channel central to cardiomyocyte biology. RYR2 Ca²⁺ release has a well-established role in activating cardiomyocyte motor proteins during excitation-contraction coupling and is therefore critical for heart function. RYR2 is also poised to have other important cardiac functions such as setting heart rate, stimulating ATP metabolism, regulating cardiac hypertrophy, and controlling cardiomyocyte survival. In addition, there is evidence that RYR2 dysfunction occurs during heart disease, suggesting that RYR2 may be a driver of cardiac pathology. The research in this thesis seeks to test which aspects of cardiomyocyte biology are regulated by RYR2 signaling and whether RYR2 loss-of-function is pathogenic. Using a heart-specific, inducible gene deletion system in mice we were able to show that loss of Ryr2 caused heart failure and reduced cardiac contraction. In addition, we saw that Ryr2 deletion lead to reduced heart rate, tachycardic arrhythmia, diminished oxidative metabolism, increased cardiac hypertrophy, and increased cell death via a novel mechanism. To test whether the metabolic and heart rate effects persist in the absence of heart failure, we used an inducible, heart-specific 50% Ryr2 deletion model. In this context we did not see heart failure or decreased cardiac function, but still observed a decrease in heart rate and altered oxidative metabolism. Unlike complete Ryr2 knockout, the 50% Ryr2 ablation model did not display a general decrease in oxidative ATP metabolism, but instead a specific decrease in glucose oxidation. This was associated with reduced mitochondrial Ca²⁺ uptake and decreased activation of the pyruvate dehydrogenase complex, a Ca²⁺ sensitive gatekeeper of glucose oxidation. Collectively, these results provide compelling evidence that RYR2 is an essential component of excitation-contraction coupling and a critical driver of cardiac pacemaking. These results also demonstrate that RYR2 is critical for mitochondrial Ca²⁺ uptake and stimulating oxidative metabolism and strongly suggest that RYR2 has a specific role in activating glucose oxidation. This research also shows that loss of Ryr2 recapitulates heart failure and suggests RYR2 may be involved in hypertrophy and cell death. This suggests a model where RYR2 simultaneously regulates a several facets of cardiomyocyte biology.Medicine, Faculty ofGraduat

Generation and characterization of a mouse model harboring the exon-3 deletion in the cardiac ryanodine receptor.

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/-) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/- mice are not susceptible to CPVT. Ex3-del+/- cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/- mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/- mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/- mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion

Generation and characterization of a mouse model harboring the exon-3 deletion in the cardiac ryanodine receptor.

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/-) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/- mice are not susceptible to CPVT. Ex3-del+/- cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/- mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/- mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/- mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion

The mitochondrial calcium uniporter underlies metabolic fuel preference in skeletal muscle

The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle-specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism

Deletion of exon-3 in the RyR2 mRNA from heterozygous RyR2 Ex3-del mice.

<p>A fragment of the mouse RyR2 mRNA covering exon-3 was converted to cDNA and amplified using RT-PCR from total RNAs isolated from wild type (WT) and heterozygous RyR2 Ex3-del (Het) mutant mice (A). The RT-PCR products were isolated and sequenced. The sequence of the RyR2 Ex3-del cDNA was shown (B). Note that the exon-2 sequence is directly followed by the exon-4 sequence, i.e. the exon-3 sequence has been deleted.</p

Reduced RyR2 protein expression in heterozygous RyR2 Ex3-del mutant hearts.

<p>(A) Whole heart homogenates were prepared from wild type (WT) (n = 4) and RyR2 Ex3-del^−/− mutant (n = 4) mice (2–3 months) and used for immunoblotting analysis using antibodies against RyR2 or β-actin. (B) The expression of RyR2 in the Ex3-del hearts was significantly reduced (58±3%) as compared to that in WT hearts (*<i>P</i><0.001).</p

Heterozygous RyR2 Ex3-del mutant mice with cardiac specific, conditional KO of the WT RyR2 allele exhibit bradycardia, but no CPVT.

<p>iRyR2^flox/flox (n = 11) and iRyR2^flox/Ex3-del (n = 8) mice were treated with tamoxifen. ECG recording was performed 12 days post tamoxifen treatment to determine their basal heart rates (before epinephrine/caffeine challenge) (A) and their susceptibility to CPVT (B, C). Representative ECG recordings of the tamoxifen-treated iRyR2^flox/flox mice (B) and the tamoxifen-treated iRyR2^flox/Ex3-del mice (C) before (top panel) and after (bottom panel) the injection of epinephrine (1.6 mg/kg) and caffeine (120 mg/kg). Note that no VTs were detected in either the tamoxifen-treated iRyR2^flox/flox mice (B) or the tamoxifen-treated iRyR2^flox/Ex3-del mice during the 30-min period of ECG recording after the injection of the triggers (*P<0.05).</p

Depolarization-induced Ca²⁺ transients in WT and heterozygous RyR2 Ex3-del mutant cardiomyocytes.

<p>Ventricular myocytes isolated from RyR2 WT and Ex3-del<b>^+/</b>⁻ mutant hearts were loaded with Rhod-2-AM and perfused with 2 mM extracellular Ca²⁺ in KRH solution and paced at 3Hz. Ca²⁺ transients were monitored by line-scan confocal Ca²⁺ imaging. Representative images/traces of WT (A) and Ex3-del<b>^+/</b>⁻ mutant (B) cardiomyocytes, and average data of the amplitude (C), time to peak (D), and time to 50% decay (E) of Ca²⁺ transients in WT and Ex3-del<b>^+/</b>⁻ mutant cells are shown. Data shown are mean ± SEM from 35 WT and 58 mutant cells (**P<0.001; *P<0.05).</p