Excess Intramyocellular Lipid Does Not Affect Muscle Fiber Biophysical Properties in Mice or People With Metabolically Abnormal Obesity.

Observational studies have shown correlations between intramyocellular lipid (IMCL) content and muscle strength and contractile function in people with metabolically abnormal obesity. However, a clear physiologic mechanism for this association is lacking, and causation is debated. We combined immunofluorescent confocal imaging with force measurements on permeabilized muscle fibers from metabolically normal and metabolically abnormal mice and people with metabolically normal (defined as normal fasting plasma glucose and glucose tolerance) and metabolically abnormal (defined as prediabetes and type 2 diabetes) overweight/obesity to evaluate relationships among myocellular lipid droplet characteristics (droplet size and density) and biophysical (active contractile and passive viscoelastic) properties. The fiber type specificity of lipid droplet parameters varied by metabolic status and by species. It was different between mice and people across the board and different between people of different metabolic status. However, despite considerable quantities of IMCL in the metabolically abnormal groups, there were no significant differences in peak active tension or passive viscoelasticity between the metabolically abnormal and control groups in mice or people. Additionally, there were no significant relationships among IMCL parameters and biophysical variables. Thus, we conclude that IMCL accumulation per se does not impact muscle fiber biophysical properties or physically impede contraction.

Insights into posttranslational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ∼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ∼65% reduction in in vivo ankle dorsiflexion torque, as compared with wild type (WT; i.e., Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume, or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.NEW & NOTEWORTHY The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both E1A binding protein p300 (p300) and cAMP-response element-binding protein binding protein (CBP) in skeletal muscle remains unknown. Here, we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca2+ and bypasses membrane depolarization.

Insights into post-translational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and thus could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ∼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ∼65% reduction in in vivo ankle dorsiflexion torque, as compared to wildtype (WT; i.e. Cre negative) littermates. Despite the profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca 2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca 2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice. New & Noteworthy: The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both p300 and CBP in skeletal muscle remains unknown. Here we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca 2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca 2+ and bypasses membrane depolarization.

A maternal high-fat, high-sucrose diet induces transgenerational cardiac mitochondrial dysfunction independently of maternal mitochondrial inheritance.

Maternal obesity is correlated with cardiovascular disease in offspring, with a 1.3-fold increase in events observed in offspring of obese women. We have observed that obesity-exposed oocytes demonstrate impaired mitophagy and transmit damaged mitochondria to the offspring. Accordingly, we hypothesized that maternal obesity induces cardiac mitochondrial dysfunction in the offspring via transgenerational inheritance of abnormal oocyte mitochondria. We mated female mice fed a high-fat/high-sucrose (HFS) diet (or chow) with chow-fed males and assessed cardiac structure and function in their descendants that were chow fed in each generation. All F1 to F3 descendants bred via the female in each generation were nonobese and demonstrated cardiac mitochondrial abnormalities with crystal rarefaction and reduced oxygen consumption pointing to a transgenerational effect, while obese F0 dams' hearts were unaffected. Furthermore, male offspring from F1 to F3 generations and female F1 and F2 offspring developed increased left ventricular (LV) mass (vs. chow-fed controls). Increased LV mass was also observed in offspring generated by in vitro fertilization of obesity-exposed oocytes and gestation in nonobese surrogates, ruling out a gestational environment effect. Contrary to our hypothesis, male F1 also transmitted these effects to their offspring, ruling out maternal mitochondria as the primary mode of transmission. We conclude that transmission of obesity-induced effects in the oocyte nucleus rather than abnormal mitochondria underlie transgenerational inheritance of cardiac mitochondrial defects in descendants of obese females. These findings will spur exploration of epigenetic alterations in the oocyte genome as potential mechanisms whereby a family history of maternal obesity predisposes to cardiovascular disease in humans.

Constitutive activation of CaMKKα signaling is sufficient but not necessary for mTORC1 activation and growth in mouse skeletal muscle.

Skeletal muscle loading/overload stimulates the Ca²âº-activated, serine/threonine kinase Ca²âº/calmodulin-dependent protein kinase kinase-α (CaMKKα); yet to date, no studies have examined whether CaMKKα regulates muscle growth. The purpose of this study was to determine if constitutive activation of CaMKKα signaling could stimulate muscle growth and if so whether CaMKKα is essential for this process. CaMKKα signaling was selectively activated in mouse muscle via expression of a constitutively active form of CaMKKα using in vivo electroporation. After 2 wk, constitutively active CaMKKα expression increased muscle weight (~10%) and protein content (~10%), demonstrating that activation of CaMKKα signaling can stimulate muscle growth. To determine if active CaMKKα expression stimulated muscle growth via increased mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis, [³H]phenylalanine incorporation into proteins was assessed with or without the mTORC1 inhibitor rapamycin. Constitutively active CaMKKα increased protein synthesis ~60%, and this increase was prevented by rapamycin, demonstrating a critical role for mTORC1 in this process. To determine if CaMKKα is essential for growth, muscles from CaMKKα knockout mice were stimulated to hypertrophy via unilateral ablation of synergist muscles (overload). Surprisingly, compared with wild-type mice, muscles from CaMKKα knockout mice exhibited greater growth (~15%) and phosphorylation of the mTORC1 substrate 70-kDa ribosomal protein S6 kinase (Thr³89; ~50%), demonstrating that CaMKKα is not essential for overload-induced mTORC1 activation or muscle growth. Collectively, these results demonstrate that activation of CaMKKα signaling is sufficient but not necessary for activation of mTORC1 signaling and growth in mouse skeletal muscle.