Effect of Subtle Energy Transmission and Tao Calligraphy Mindfulness Practice on Mitochondrial DNA in Peripheral Blood Leukocytes

Objective

The goal of this study is to measure the effects of a unique form of Tao Calligraphy mindfulness practice that involves engaging with Tao art (tracing the Tao Calligraphy "Greatest Love" while listening to or singing the Tao Music or Tao Song "Greatest Love") together with subtle energy transmission on Mitochondrial DNA in peripheral blood leucocytes.

In our previous study, the participants who regularly practiced, shown an increase of the length in telomeres in peripheral blood granulocytes. In our other studies, participants reported a decrease in symptoms of their illness as perceived subjectively, improved signs reported by treating clinicians and an improvement of well-being as measured by standardized scientific questionnaires.

Hypotheses

The research null hypothesis is that individuals who receive subtle energy transmission for mitochondrial DNA in hemato-lymphoid tissue, and who will engage in daily Tao Calligraphy mindfulness practice for 12 months, will have no significant change in Mitochondrial DNA content in peripheral blood leucocytes in follow-up analyses and will show no improvement of well-being as measured by standardized scientific questionnaire Rand SF-36 at 3, 6 and 12 months.

For statistical analysis of the data from laboratory assessment and scores obtained from questionnaires, the Anova, T-Test and regression analysis will be used to evaluate the null hypothesis. The p value will represent how unlikely the observed data would be if the null hypothesis were actually true and investigators will use them to reach conclusions. The confidence level is set at 95% and if we receive p \< 0.05, then H0 is rejected. The correlation coefficient will be used to determine any correlation between various factors (e.g. effects of age, sex, length, and frequency of mindfulness practices and other) on outcome and regression analysis will be used to determine the relation of independent and dependent variables.

Tools such as the Minitabs version 14 and or Statistical Package for the Social Sciences (SPSS) or Free version of Statistical Package for the Social Sciences (PSPP) will be used.

Background and Theoretical Framework

Mitochondria are membrane bound, self-replicating organelles present in almost all eukaryotic cells. Mitochondrial energy production is essential for all cellular processes. In general, each human cell contains several hundred to 1,000 mitochondria, each with 2-10 copies of Mitochondrial DNA encoding 13 proteins essential for respiratory chain function. Thought to have originated from symbiotic ancestors, mitochondria contribute to many processes central to cellular function and dysfunction including calcium signalling, cell growth and differentiation, cell cycle control and cell death. Mitochondrial shape and positioning in cells is tightly regulated by processes of fission and fusion, biogenesis and autophagy, ensuring a relatively constant mitochondrial population. Mitochondrial inheritance is generally accepted to be maternal although small amounts of paternally transmitted mitochondria have been discovered on rare occasions.

Mitochondrial dysfunction is considered one of the hallmarks of aging and age-associated diseases. The mitochondrial genome is more vulnerable to oxidative damage and undergoes a higher rate of mutation compared with nuclear DNA. Additional underlying causes of mitochondrial dysfunction include inadequate mitochondrial quantity, exposure to environmental toxins, reduction in mitochondrial membrane permeability, impaired transport of essential metabolites, and malfunctioning of the electron transport chain and Adenosine triphosphate (ATP) synthesis.

Unfortunately, no measure is currently available that globally assesses the ability of mitochondria to perform normal biological functions. Mitochondrial functions include but are not limited to oxidative phosphorylation and energy production in the form of Adenosine triphosphate (ATP) , reactive oxygen species (ROS) production, cell death signalling, as well as steroid hormone synthesis, and systemic signalling.

It has been proposed that the number of Mitochondrial DNA copies per cell (Mitochondrial DNA copy number) reflects mitochondrial health. A major driver of the popularity of Mitochondrial DNA copy number as a potential marker of mitochondrial health lies in its ease of measure from stored DNA, or indirectly from genotyping/sequencing data. Compared to direct assays of mitochondrial function, which require fresh tissue, the scalability of Mitochondrial DNA copy number assessments is appealing for biomarker studies.

However, Mitochondrial DNA copy number does not directly reflect respiratory chain (RC) function or energy production capacity. According to the theory of "biochemical threshold", only when Mitochondrial DNA copy number decreases by 60 to 80% of normal levels does RC function and energy production capacity decrease. This means that in many cases 20-40% of the baseline Mitochondrial DNA copy number is sufficient to produce the 13 proteins necessary to sustain respiratory capacity. But this level of Mitochondrial DNA depletion seems to occur only in rare mitochondrial diseases or in isolated single cells in diseased organs. The uncoupling of Mitochondrial DNA copy number and respiratory capacity may be accounted for by the fact that up-regulation of transcription and translation from pre-existing Mitochondrial DNA copies can increase the levels of messenger ribonucleic acid (mRNA), protein subunits, RC function, and energy production capacity, without a change in Mitochondrial DNA copy number. This notably occurs in response to exercise, where mitochondrial content and RC activity in human muscle increases within days to weeks without a change in Mitochondrial DNA copy number. Thus, in human tissues, Mitochondrial DNA copy number is not directly coupled to, and does not directly reflect mitochondrial bioenergetics.

Furthermore, the picture is somewhat complicated in that while low blood Mitochondrial DNA copy number has been associated with neurodegenerative disease, cardiovascular disease, and both cognitive and physical performance in aging; other conditions such as diabetes, major depression, some cancers, and mitochondrial disorders are associated with elevated Mitochondrial DNA copy number. Regarding exposure to environmental toxicants, Mitochondrial DNA copy number has been associated with either higher or lower Mitochondrial DNA copy number.

Nevertheless, the value and specificity of blood Mitochondrial DNA copy number can be increased in several ways as summarized below:

1. Use data from complete blood counts with differential, enhanced using flow cytometry methods that quantify immunologically-defined cell subpopulations, then analyses with multivariate models (i.e., deconvolution method) to understand the proportion of variance in Mitochondrial DNA copy number attributable not just to general immune cell categories (e.g., granulocytes and lymphocytes) but to specific cell subtypes (naive and memory cluster of differentiation 4 (CD4) and cluster of differentiation 8 (CD8) T cells, B cells, subtypes of monocytes, etc). 2. Further enhance the sensitivity and interpretability of Mitochondrial DNA copy number in relation to health related phenotypes by quantifying Mitochondrial DNA copy number directly in molecularly-defined subtypes of immune cells. For example, CD4+ Naive T cells, monocytes, or other specific immune subtype exist in sufficient abundance in circulation to be isolated by either flow cytometric cell sorting (also known as fluorescence-activated cell sorting, FACS) or by negative/positive selection by magnetic activated cell sorting. Compared to cell mixtures, cell-specific Mitochondrial DNA copy number quantification add biological specificity to detect meaningful mitochondrial associations related to exposures, other biomarkers, and possibly age and sex-related differences. 3. Measure other markers of mitochondrial content and/or function in parallel with Mitochondrial DNA copy number, e.g. live assays of mitochondrial function. Some examples include i) citrate synthase (CS) activity, cardiolipin, or mitochondrial protein abundance to estimate mitochondrial content; ii) Mitochondrial DNA integrity, such as DNA damage, point mutations, or deletions; or iii) mitochondrial respiratory capacity, such as oxygen consumption by respirometry, which can and should be performed in specific cell types, as well as respiratory chain enzymatic activities that reflect energy production capacity on either a per-cell or per-mitochondrion basis. In the context of direct measurements of respiratory chain function, Mitochondrial DNA copy number becomes a more biologically interpretable feature of mitochondrial health. 4. Measure circulating markers of mitochondrial stress accessible in plasma or other bio fluids, including Growth Differentiation Factor 15 (GDF15), cf Mitochondrial DNA or other emerging mitochondrial kinesis. However, these may lack specificity. E.g. GDF15, cf Mitochondrial DNA, and other circulating markers can be induced by a number of stressors not necessarily reflecting mitochondrial RC capacity or stress. 5. There is a negative correlation between the copy number of Mitochondrial DNA, number of mutations in mitochondrial deoxyribonucleic acid (Mitochondrial DNA) and longevity. Higher rates of somatic Mitochondrial DNA mutations are consistently associated with aging phenotypes, increased risk of age-related diseases, and reduced lifespan in both animal models and human populations. 6. Whole Mitochondrial DNA sequencing (via next-generation sequencing or Sanger sequencing) can identify single nucleotide polymorphisms (SNPs), haplogroups, and mutational burden associated with longevity. This approach enables detection of specific SNPs and haplogroups that have been associated with increased lifespan. 7. The mitochondrial-to-nuclear genome ratio (Mitochondrial DNA content) in tissues and body fluids correlates with the size and number of mitochondria. Polymerase Chain Reaction (PCR) based molecular test to determine mitochondrial-to-nuclear genome ratio in peripheral blood leukocytes.

It has been concluded that although the biological interpretation of differences in Mitochondrial DNA copy number is tenuous, in combination with relevant markers assessed in homogenous or well-defined cell populations, continuing to add Mitochondrial DNA copy number to existing studies with rich sets of outcomes is likely to contribute valuable insights into the role of mitochondria in human health, aging, and resilience.

Mindfulness Practices and Mitochondria

Psychologically, mindfulness reduces cognitive and emotional reactivity, rumination, and worry, while increasing self-awareness and acceptance, which mediate improvements in mental health and facilitate healthier behaviours. These psychological changes are tightly linked to the observed biological effects.

The biological mechanisms by which mindfulness practices improve health involve down-regulation of stress and inflammatory pathways, enhancement of immune function, and neoplastic changes supporting self-regulation and adaptive coping.

Mindfulness practices may contribute to prolonging human life by improving mitochondrial function, reducing cellular stress, and promoting adaptive stress responses that enhance cellular maintenance and resilience. Chronic psychological stress accelerates mitochondrial dysfunction, increases oxidative damage, and impairs cellular repair mechanisms, all of which are associated with aging and reduced lifespan. Mindfulness-based interventions have been shown to mitigate these effects by reducing stress-induced inflammation and oxidative stress, thereby supporting mitochondrial health.

Mitochondria regulate key longevity pathways through energy production, redox signalling, and mitochondrial-nuclear communication. Mindfulness practices can ac

• Mean Change from Baseline in Quality of Life Scores on 36-item short-form Quality of Life Questionnaire (SF-36) at 3 months, 6 months and 12 months [Time frame: From start of Mindfulness practices with Tao Art (Calligraphy and Song Greatest Love) to end of practices at 12 months.]