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Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca 2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition. Little is known about how cardiac metabolism remodels following cardiac injury. Here, the authors show that mitochondrial CaMKII plays an important role in remodeling cardiac metabolism after injury and that replacement of mitochondrial creatine kinase improves energetics and protects against adverse remodeling.

Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.

Excitation-contraction (E-C) coupling is fundamental to heart contraction. Junctophilin-2 is a structural protein required for formation of the E-C coupling machinery. During heart disease, stress-activated calpain cleaves junctophilin-2, disrupting the E-C coupling machinery and calcium ion signaling, which compromises cell contraction. Guo et al. found that under stress conditions, calpain-mediated cleavage converted full-length junctophilin-2 from a structural protein into a transcriptional regulator that shuttled to the nucleus (see the Perspective by Padmanabhan and Haldar). Furthermore, failing cardiomyocytes in stressed myocardium transduced mechanical information (E-C uncoupling) into transcriptional reprogramming. Science , this issue p. eaan3303 ; see also p. 1359 A protein involved in excitation-contraction coupling regulates transcription and helps to protect cardiac tissues from stress. Junctophilin-2 (JP2) is a structural protein required for normal excitation-contraction (E-C) coupling. After cardiac stress, JP2 is cleaved by the calcium ion–dependent protease calpain, which disrupts the E-C coupling ultrastructural machinery and drives heart failure progression. We found that stress-induced proteolysis of JP2 liberates an N-terminal fragment (JP2NT) that translocates to the nucleus, binds to genomic DNA, and controls expression of a spectrum of genes in cardiomyocytes. Transgenic overexpression of JP2NT in mice modifies the transcriptional profile, resulting in attenuated pathological remodeling in response to cardiac stress. Conversely, loss of nuclear JP2NT function accelerates stress-induced development of hypertrophy and heart failure in mutant mice. These data reveal a self-protective mechanism in failing cardiomyocytes that transduce mechanical information (E-C uncoupling) into salutary transcriptional reprogramming in the stressed heart.

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

The h yperpolarization-activated c yclic n ucleotide-gated ion channel 4 (HCN4) current increases due to cAMP binding and is well-recognized to contribute to adrenergically driven heart rate acceleration. HCN4 current also increases with heat by an unknown mechanism(s). We use thermodynamical and homology computational modeling, site-directed mutagenesis, and mouse models to identify a concise motif on the S4-S5 linker of HCN4 channels (M407/Y409) that determines HCN4 current (I f ) responses to heat. This motif is required for heat-triggered rate acceleration in cardiac pacemaker cells, isolated hearts and in vivo. Surprisingly, a loss of function M407/Y409 motif mutation prevented not only normal heat but also cAMP responses, suggesting that the heat-sensing machinery within the S4-S5 linker is essential for operating the cAMP allosteric pathway and is central to HCN4 gating modulation. The M407/Y409 motif is conserved across all HCN family members suggesting that HCN channels participate broadly in coupling heat to changes in cell membrane excitability. Heat increases heart rate across vertebrates. Here, authors pinpoint a motif in the HCN4 channel that governs heat-driven heart rate acceleration and demonstrate its conserved role in other HCN channels, revealing a conserved mechanism linking temperature to membrane excitability.

Myocardial mitochondrial Ca ²⁺ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca ²⁺ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca ²⁺ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca ²⁺ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O ₂ consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU–permeabilized myocardial fibers or isolated mitochondria in low Ca ²⁺ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca ²⁺ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca ²⁺] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca ²⁺ homeostasis. Mitochondrial Ca ²⁺ overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca ²⁺ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury. Mitochondrial Ca ²⁺ is a fundamental signal that allows for adaptation to physiological stress but a liability during ischemia-reperfusion injury in heart. On one hand, mitochondrial Ca ²⁺ entry coordinates energy supply and demand in myocardium by increasing the activity of matrix dehydrogenases to augment ATP production by oxidative phosphorylation. On the other hand, inhibiting mitochondrial Ca ²⁺ overload is promulgated as a therapeutic approach to preserve myocardial tissue following ischemia-reperfusion injury. We developed a new mouse model of myocardial-targeted transgenic dominant-negative mitochondrial Ca ²⁺ uniporter (MCU) expression to test consequences of chronic loss of MCU-mediated Ca ²⁺ entry in heart. Here we show that MCU inhibition has unanticipated consequences on extramitochondrial pathways affecting oxygen utilization, cytoplasmic Ca ²⁺ homeostasis, physiologic responses to stress, and pathologic responses to ischemia-reperfusion injury.

Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant-negative (DN) MCU. Here, we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found that MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU-enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment before each heartbeat. Our findings show that MCU is necessary for complete physiological heart rate acceleration and suggest that MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate. Animals react to threats by increasing their heart rate. Wu et al . show that mitochondrial calcium uptake via a highly selective ion channel, the mitochondrial calcium uniporter, stimulates metabolism in cardiac pacemaker cells and is essential for physiological pulse acceleration but not resting heart rate.