Early-Life Persistent Vitamin D Deficiency Alters Cardiopulmonary Responses to Particulate Matter-Enhanced Atmospheric Smog in Adult Mice

Abstract

Early-life nutritional deficiencies can lead to increased cardiovascular susceptibility to environmental exposures. Thus, the purpose of the study was to examine the effect of early-life persistent vitamin D deficiency (VDD) on the cardiopulmonary response to a particulate matter-enhanced photochemical smog. Mice were fed a VDD or normal diet (ND) after weaning. At 17 weeks of age, mice were implanted with radiotelemeters to monitor electrocardiogram, heart rate (HR) and heart rate variability (HRV). Ventilatory function was measured throughout the diet, before and after smog exposure using whole-body plethysmography. VDD mice had lower HR, increased HRV and decreased tidal volume compared with ND. Regardless of diet, HR decreased during air exposure; this response was blunted by smog in ND mice and to a lesser degree in VDD. When compared with ND, VDD increased HRV during air exposure and more so with smog. However, smog only increased cardiac arrhythmias in ND mice. This study demonstrates that VDD alters the cardiopulmonary response to smog highlighting the possible influence of nutritional factors in determining responses to air pollution. The mechanism of how VDD induces these effects is currently unknown, but modifiable factors should be considered when performing risk assessment of complex air pollution atmospheres.

Graphical Abstract

Introduction

It is clear from epidemiological, human and animal studies that air pollution has a deleterious effect on cardiovascular health (e.g. ischemic heart disease, arrhythmia, stroke)¹. In fact, research from the last ten years has shown that there are several factors, which include not only those related to air pollution such as concentration, composition and chemistry, but also host factors like nutrition, that contribute to the overall response. Thus, characterization of these factors with the intent of understanding the underlying toxicological mechanisms as well as providing useable public health information about individual susceptibility is crucial to reducing the harmful effects of air pollution particularly given the prevalence of chronic diseases like asthma and heart disease.

Like all the organ systems, regulation and proper function of the cardiovascular system is dependent on adequate levels of micronutrients like vitamin D, which is one of a few molecules with a critical homeostatic role throughout the body.^{2, 3} Although recent data demonstrate an association between vitamin D deficiency (VDD) and cardiovascular impairment^4–6, the link to disease development is still not firmly established, nor is it clear whether it contributes to adverse responses due to air pollution. Vitamin D is a fat-soluble vitamin produced endogenously in the skin, after ultraviolent B radiation exposure, and acquired through the diet from a variety of foods like milk, fish, cheese and beans.^{2, 7} Vitamin D receptors are present on numerous tissues and cells throughout the body, including cardiomyocytes.² Yet, minimal sun exposure, obesity, improper nutrition as well as a myriad of other factors result in VDD^{2, 3}, which has become a public health concern affecting 8% of the pediatric population in the United States.⁸ Early-life or childhood VDD can lead to vascular dysfunction, hypertension and other cardiac abnormalities^9–11, although its precise role in electrocardiographic abnormalities and cardiac autonomic changes has not been extensively characterized. Vitamin D exerts its effects through a steroid nuclear receptor ^{2, 12} that binds to vitamin D response elements (VDRE) on DNA and influences expression of a wide variety of genes affecting the function of numerous organ systems, including the cardiovascular.^12–15,16

In relation, chronic VDD may cause subtle latent physiological changes which increase the risk of a triggered adverse response to a stressor like air pollution. These adverse responses triggered by exposure are far more difficult to characterize with complex, particulate matter (PM)-rich photochemical smog, which is comprised of not only PM but also ozone and other gaseous pollutants (e.g. acrolein and aldehyde isoforms), and which represents the bulk of what people are exposed to in terms of ambient air pollution.^17–19 The purpose of this study was to determine the effect of early-life persistent VDD in smog-induced cardiovascular toxicity. We hypothesized that VDD during early-life and persisting into adulthood would 1) modulate autonomic influence on the heart and induce electrical changes, 2) cause ventilatory alterations, and as a result 3) alter the cardiopulmonary response to atmospheric smog.

Materials and Methods

Animals -

Three-week old female C57Bl/6 mice (body weight = 9.6 ± 1.6g) were used in this study (Jackson Laboratory – Raleigh, NC). Mice were housed five per cage and maintained on a 12-hr light/dark cycle at approximately 22˚C and 50% relative humidity in an AAA-LAC-approved facility. Food (Prolab RMH 3000; PMI Nutrition International, St. Louis, MO) and water were provided ad libitum during the quarantine period (3 days) after arrival. All protocols were approved by the Institutional Animal Care and Use Committee of the U.S. Environmental Protection Agency and are in accordance with the National Institutes of Health Guides for the Care and Use of Laboratory Animals. The animals were treated humanely and with regard for alleviation of suffering.

Diet –

Mice were randomly placed and maintained ad libitum on either a VDD (D10073001) or normal diet (ND) (D10012G-Research Diets Inc) for 16 weeks. The VDD diet had no added vitamin D. The ND has 1000 IU per 10 grams of vitamin D. The diets had equal levels of all other vitamins and minerals including calcium, which was at the concentration specified by the American Institute of Nutrition.²⁰

Experimental Groups –

Mice were randomly assigned into a ND (n = 28) or VDD (n = 35) group and maintained on those diets for the extent of the study. Of those animals, 12 of each diet were randomly chosen and implanted with radiotelemeters at 16 weeks of age. Each of the groups with radiotelemeters were then further randomly assigned to air (FA) or smog (SA-PM) exposure groups as were the non-telemetered ND and VDD mice. The timeline of the experimental design is depicted in Figure S1.

Surgical Implantation of radiotelemeters and data acquisition –

Animals were implanted with radiotelemeters and monitored as previously described²¹. Briefly, animals were anesthetized using inhaled isoflurane (Butler Animal Health Supply, Dublin OH) and using aseptic technique, each animal was implanted subcutaneously with a radiotelemeter (ETA-F10, Data Sciences International, St Paul, MN) to approximate the lead II of a standard electrocardiogram (ECG). Signals from the radiotelemeters were used to monitor heart rate (HR) and ECG waveforms immediately following telemeter implantation, through exposure until 24hrs post-exposure. This methodology provided continuous monitoring and collection of physiologic data from individual mice. See Supporting Information for specific details.

Heart Rate Variability Analysis -

Heart rate variability (HRV) was calculated as the mean of the differences between sequential RRs for the complete set of ECG waveforms. For each 1-min stream of ECG waveforms, mean time between successive QRS complex peaks (RR interval), mean HR, and mean HRV-analysis–generated time-domain measures were acquired. The time-domain measures included standard deviation of the time between normal-to-normal beats (SDNN), and root mean squared of successive differences (RMSSD). HRV analysis was also conducted in the frequency domain using a Fast-Fourier transform. The spectral power obtained from this transformation represents the total harmonic variability for the frequency range being analyzed. In this study, the spectrum was divided into low-frequency (LF) and high-frequency (HF) regions. The ratio of these two frequency domains (LF/HF) provides an estimate of the relative balance between sympathetic (LF) and vagal (HF) activity.

Whole-Body Plethysmography -

Ventilatory function (e.g. enhanced pause, tidal volume and minute ventilation) was assessed in awake, unrestrained mice using a whole-body plethysmograph (Buxco, Wilmington, NC). Assessments were performed at 3, 8, 11 and 15-weeks of age and 24hrs prior to the day of exposure, immediately post-exposure and 24hrs after exposure. Using respiratory-induced fluctuations in ambient pressure, ventilatory parameters including tidal volume (VT), breathing frequency (f), inspiratory time (Ti), expiratory time (Te), minute volume (MV) and enhanced pause (Penh), which is a measure of ventilatory timing and can indicate airway irritation, were calculated and recorded on a breath-by-breath basis.

Tissue Collection and Analysis -

Mice were euthanized 24 hours after exposure and blood was collected, processed and analyzed. Vitamin D concentrations were determined in the serum spectrophotometrically using a Vitamin D EIA Kit (Cayman Chemical, Ann Arbor, Michigan).

Photochemical Smog Exposures –

An atmosphere with high particulate matter (PM) and low ozone and nitrogen oxide concentrations (SA-PM) was generated in the Mobile Reaction Chamber (MRC). Briefly, SA-PM was artificially generated with 0.491 ppm nitrogen oxide (NO), 0.528 ppm NOx, 29.9 ppmC total hydrocarbons (THC), 24 ppmC gasoline and 5.3 ppmC α -pinene as the initial conditions, which were then irradiated by ultraviolent light. SA-PM was transported under vacuum to 0.3 m⁻³ whole body inhalation chambers. Continuous gas and aerosol sampling for carbon monoxide (CO), ozone (O₃), nitrogen oxides (NOx), THC and particle mass concentration were conducted at both the MRC unit as well as from the inhalation exposure systems. All PM was formed as secondary organic aerosol (SOA) from the photochemical reactions in the MRC. Particle size distributions and gravimetric mass sampling was measured. Filter sampling for gravimetric analysis were conducted for the entire exposure time. Volatile organic compound (VOC) summa cannisters were periodically collected and analyzed by gas chromatography off-line to determine concentrations of various VOCs in the exposure atmosphere. Please refer to Krug et al. in this issue for complete exposure details.

Statistics -

All data were analyzed using SAS 9.4 (SAS Institute Inc., Cary, NC) software. Mixed-model ANOVAs, with Tukey’s procedure for the post hoc comparisons, were used to examine the statistical differences between exposure and diet. To improve normality of the residuals and because the HRV variable distributions were highly skewed, each HRV parameter was natural- log transformed. Also, the delta values of the variables from baseline were used in this analysis. The statistical significance was set at P < 0.05.

Results

Exposure characteristics -

The measured criteria pollutants in SA-PM (MR044) were ozone (0.094 ppm), NO_x (0.154 ppb) and PM_2.5 (0.307 mg/m³). The ten most abundant secondary compounds that were generated in the atmosphere were: ethanol (1.03 ppm), alpha-pinene (0.533 ppm), toluene (0.463 ppm), 2-methylpentane (0.330 ppm), n-hexane (0.222 ppm), isopentane (0.212 ppm), m-& p-xylene (0.201 ppm), 3-methylpentane (0.187 ppm), n-pentane (0.103 ppm) and n-butane (0.0748 ppm) (Figure S2). See Krug et al. in this issue for further details.

Body weight and vitamin D levels -

Body weight was determined on a weekly basis; there were no differences between ND and VDD mice (Figure S3). Vitamin D levels were confirmed to be lower in all VDD mice (1.06 ± 0.1 ng/mL) when compared with ND mice (21.1±2.2 ng/mL), but there was no difference in calcium or phosphate levels (Data not shown).

Heart rate and heart rate variability –

HR was decreased in VDD mice from the time of telemeter implantation until exposure (Figure 1A), while SDNN, RMSSD, and HF increased (Figure 1B–D). Log-normal distribution was calculated for HRV because the data were not normally distributed. HR decreased significantly 24 hours after FA in ND animals and after SA-PM exposure in VDD animals. ND animals exposed to SA-PM and VDD animals exposed to air did not exhibit any changes (i.e. their HR did not decrease after exposure) (Figure 2A). SDNN was increased in all groups 24hrs after exposure when compared to pre-exposure, yet it only decreased in the 24hrs after exposure in ND mice exposed to SA-PM. VDD increased SDNN irrespective of the exposure, yet when combined, VDD and SA-PM had a significantly greater effect on SDNN than either alone (Figure 2B). Similar trends were observed with RMSSD for which the combination of VDD and SA-PM had a greater effect than either VDD or SA-PM alone (Figure 2C). HF decreased significantly due to VDD and SA-PM exposure (Figure 2D); interestingly, this phenomenon was also observed in ND mice exposed to FA but not in VDD mice exposed to FA or the ND mice exposed to SA-PM. No significant changes were observed in LF (data not shown).

Figure 1.

VDD mice had decreased heart rate and increased HRV prior to exposure. (A.) Heart rate was significantly decreased in VDD mice when compared to ND, whereas SDNN (B.), RMSSD (C.), and HF (D.) were significantly increased. Parameters were analyzed from the time of radiotelemeter implantation until exposure. ^◊Denotes a significant change from ND (p < 0.05). Values represent means ± SE.

Figure 2.

VDD alters heart rate and heart rate variability after SA-PM exposure. There were no changes in HR or HRV immediately after exposure in both ND and VDD mice (A - D). SDNN was increased in all groups 24hrs after exposure when compared to pre-exposure except ND mice exposed to SA-PM. VDD increased SDNN irrespective of the exposure, yet when combined, VDD and SA-PM had a significantly greater effect on SDNN than either alone (B.). Similar trends were observed with RMSSD for which the combination of VDD and SA-PM had a greater effect than either VDD or SA-PM alone (C.). HF decreased significantly in VDD mice exposed to SA-PM and ND mice exposed to FA but not in VDD mice exposed to FA or ND mice exposed to SA-PM (D.) *Denotes a significant change from pre-exposure (p < 0.05). ^◊Denotes a significant change from ND (p < 0.05). ^†Denotes a significant change from immediately post-exposure. ^‡Denotes a significant change from filtered air exposure. Values represent means ± SE.

During exposure, HR decreased from baseline (30-minute acclimation period before exposure) in all animals regardless of diet or exposure. SA-PM significantly blunted the decrease in HR in ND mice; a similar effect was seen in VDD mice but to a lesser degree (Figure 3A). SDNN increased in VDD mice during air exposure and in ND mice during smog exposure however there was no effect in VDD mice exposed to SA-PM (Figure 3B). In addition, RMSSD increased significantly in VDD mice during air exposure when compared with ND; this response was further increased if the animals were exposed to SA-PM (Figure 3C). HF only increased in VDD mice during SA-PM exposure (Figure 3D). No significant changes were observed in LF during exposure. Lastly, the number of arrhythmias significantly increased in ND mice during SA-PM exposure and although VDD appears to also increase total arrhythmias the results were not significant (Figure 3E).

Figure 3.

Exposure to SA-PM prevents the recovery of resting heart rate in normal and VDD mice but only potentiates parasympathetic modulation in the latter. (A.) SA-PM significantly blunted the decrease in HR in both ND and VDD mice. (B.) SDNN increased in VDD mice during FA and in ND mice during SA-PM exposure, however there was no effect in VDD mice exposed to SA-PM. (C.) RMSSD increased significantly in VDD mice during FA when compared to ND; this response was further increased with SA-PM. (D.) VDD mice exposed to SA-PM had significantly increased HF when compared to ND and VDD mice exposed to FA. (E.) VDD mice exposed to air had significantly increased arrhythmia when compared to SA-PM, but there was no effect of SA-PM in VDD mice. *Denotes a significant change from baseline of exposure (p < 0.05). ^◊Denotes a significant change from ND (p < 0.05). ^‡Denotes a significant change from filtered air exposure (p < 0.05). Values represent means ± SE.

Ventilatory function –

Although ND mice had lower f at 3 weeks of age than VDD, the f was comparable at 8 weeks and continued to decrease with age for both groups (Figure 4A). Similarly, although V_T/MV were higher in the VDD group at the beginning of the diet further increases in V_T/MV with aging over time were blunted in the VDD mice when compared with ND mice (Figure 4B – VDD 72.4% increase; ND 111.2% increase at 15wks). VDD mice had decreased Ti before and through the diet regimen when compared with ND mice while there were no differences in Te (Table S1).

Figure 4.

VDD mice have impaired development of normal breathing patterns and higher breathing frequency during SA-PM exposure. VDD mice had significantly less decrease in f and increase in Vt during the 15 weeks of development when compared to ND (A. and B.). SA-PM prevented f and penh from decreasing in both ND and VDD, but VDD mice had a significantly greater effect than ND (C. and D.). *Denotes a significant change from 3-week assessment (p < 0.05). ^◊Denotes a significant change from ND (p < 0.05). C-D. The percent change from pre-exposure. ^‡Denotes a significant change from filtered air exposure (p < 0.05). ^◊Denotes a significant change from ND (p < 0.05). Values represent means ± SE.

Other than ventilatory timing (penh) being higher in VDD versus ND mice, there were no significant differences in the pre-exposure ventilatory parameters between the two diets (Table 1). All subsequent comparisons for exposure-related ventilatory effects were made within group. As such, all animals experienced a significant decrease in f after exposure; however, this decrease was less in VDD mice when compared with ND and the effect was further reduced by SA-PM (i.e. f decreased the least in VDD mice after SA-PM). Similar trends were observed with V_T, Ti, and Te however the values increased after exposure. Penh increased after SA-PM exposure in ND mice when compared to pre-exposure and with respect to their controls; all the other groups including VDD mice exposed to SA-PM had a decrease in penh post-exposure.

Table 1.

Ventilatory Function of ND and VDD Mice Is Altered after Photochemical SA-PM Exposure

diet	exposure	breathing frequency (f)	tidal volume (mL)	inspiratory time (ms)	expiratory time (ms)	minute volume (mL/min)	ventilatory timing (Penh)
ND	air	pre 437.9 ± 0.6	0.29 ± 0.0	61.89 ± 0.1	79.64 ± 0.1	12.59 ± 0.0	1.38 ± 0.0
		post410.8 ± 0.6a	0.31 ± 0.0a	66.07 ± 0.1a	86.93 ± 0.2a	12.53 ± 0.0	1.15 ± 0.0a
	SA-PM	pre 427.8 ± 0.6	0.29 ± 0.0	62.04 ± 0.1	83.73 ± 0.1	12.15 ± 0.0	1.13 ± 0.0
		post405.7 ± 0.6a	0.31 ± 0.0ac	66.08 ± 0.1a	87.93 ± 0.2ac	12.29 ± 0.0a	1.31 ± 0.0ac
VDD	air	pre 436.2 ± 0.6	0.31 ± 0.0b	61.26 ± 0.1b	81.03 ± 0.1b	12.14 ± 0.0b	1.43 ± 0.0b
		post415.6 ± 0.7ab	0.30 ± 0.0ab	64.28 ± 0.1ab	86.94 ± 0.2a	12.18 ± 0.0b	1.28 ± 0.0ab
	SA-PM	pre 434.9 ± 0.6b	0.29 ± 0.0	62.24 ± 0.1	82.93 ± 0.2b	12.33 ± 0.0b	1.65 ± 0.0b
		post424.4 ± 0.6abc	0.30 ± 0.0abc	63.38 ± 0.1abc	84.45 ± 0.2abc	12.57 ± 0.0abc	1.38 ± 0.0abc

^aDenotes a significant change from pre-exposure (p < 0.05).

^bDenotes a significant change from ND (p < 0.05).

^cDenotes a significant change from filtered air exposure (p < 0.05). Values represent means ± SE.

Electrocardiogram analysis –

There were no significant differences in any parameters during the pre-exposure period. The PR interval decreased significantly from baseline during air exposure in both ND and VDD animals but there were no differences between the diets during, immediately and 24hrs after exposure. In contrast, the PR interval of VDD mice was increased compared with ND mice during SA-PM exposure, immediately after and 24hrs later. SA-PM exposure caused a decreased PR interval in ND mice and increased PR interval in VDD mice when compared to their respective air-exposed controls. VDD animals had a decreased QRS interval during air exposure when compared to ND; this difference between VDD and ND was also observed in animals exposed to SA-PM and persisted 24hrs after that exposure. Smog exposure also caused a significant decrease in QRS complex in ND mice when compared to controls. Lastly, VDD caused a decrease in QTc when compared with ND with either air or SA-PM exposure during either exposure or immediately after but there were no significant differences among any group for the most part (Table S2).

Discussion

This study demonstrates that early life and persistent vitamin D deficiency into adulthood modifies the cardiopulmonary response to PM-enhanced smog exposure. These results add to a growing body of research which shows that the degree and quality of health effects of air pollution are not only governed by concentration and composition but also non-environmental factors. Some of these factors are directly related to the host and aspects of daily living which likely accrue intrinsic changes in the body over many years and alter the degree of responsiveness to an environmental challenge or the ability to compensate for one. Such might be the case with VDD, which has become a growing public health concern in the United States with some estimates of prevalence reaching 41.6%²². This is of concern because millions of children may be deficient⁸ and remain so into adulthood thus increasing the likelihood of chronic diseases. Although it has not been studied as extensively as bone-related maladies, the cardiopulmonary effects that result from VDD are increasingly being recognized as a cause, or promoter in the least, of several long-term diseases and heightened susceptibility to triggered adverse responses. This is the first study to show that VDD during development and into adulthood alters and potentially worsens the response to air pollution in mice.

Traditional toxicological investigations, particularly in rodents, have been using susceptible models (hypertension, metabolic syndrome, etc) to further characterize the risk of adverse responses to air pollution. Our own studies indicate that rodent strains with underlying cardiovascular disease have a worse response (e.g. arrhythmia) to air pollution than normal strains although the effects are often latent and can only be observed when a subsequent challenge or trigger is used to manifest them^23–28. This suggests that to some degree the adverse effects of air pollution may be due more to disruption of homeostasis than direct tissue toxicity. Imbalance in autonomic control of the cardiovascular system as measured by heart rate variability is an example of such changes and has been well documented not only in rodents but in humans as well^29–31. Furthermore, the importance of such an assessment rests in the fact that it indicates a subtle shift in the underlying regulation of the body’s dynamic systems which may not be manifested as clinical symptoms or overt signs of toxicity.

Although the VDD mice were maintained on the deficient diet immediately post-weaning there was no difference in the body weights or the growth of VDD animals after 15 weeks as determined by tibia length (not shown) when compared with ND. Even calcium levels were found to be in the normal range for all mice irrespective of diet. Furthermore, there were no differences in the electrocardiogram of ND and VDD mice, which suggests development of the heart in the latter was normal, at least from the perspective of electrical conduction. Despite this seemingly normal development, VDD deficient mice had increased heart rate variability, indicating altered cardiac autonomic function, when compared to controls in the two weeks prior to exposure. Although decreased HRV is generally thought of as the primary indicator of cardiac risk^{32, 33}, an increase in HRV may not necessarily be a positive sign³⁴. Instead, it may be the change from normal which suggests an impaired regulation of the cardiovascular system, rather than a direct impact on the heart itself, and in a way that may not be entirely appreciated until the body is challenged and has to maintain homeostasis. Increases in vagal tone or HRV have been associated with adverse cardiovascular events in diabetes³⁵, and linked with increased mortality in heart failure patients and the elderly^{34, 36}. In addition, increased RMSSD has been found to be associated with elevated risk of air pollution-induced arrhythmia³⁷.

An increase in HRV suggests parasympathetic modulation of the cardiovascular system which was clearly seen as a significantly lower heart rate in the VDD animals when compared with ND. Vitamin D deficiency is linked to HRV changes in humans as well^38–40 and although the profile differs from rodents the overall conclusion that subtle underlying alterations are occurring in the deficient state holds true. It is likely the differences between humans and mice may be due to the fact that in contrast to humans, baseline heart rate in mice is influenced greatly by the sympathetic branch⁴¹ and short-term HRV is under the control of parasympathetic modulation^{42, 43}. Furthermore, the HRV effects of VDD in humans may be due to other disease processes (e.g. kidney disease) related to chronic deficiency which is still not confirmed to occur in mice. It may be that mice develop a similar HRV decrement if left on the deficient diet for a longer period; our future studies will address this issue. It is not entirely clear why VDD causes altered autonomic function in mice. One study showed that VDD caused changes in calcium flux and cardiomyocyte contraction-relaxation, which was shown to be linked to vitamin D receptors (VDR) located on the T-tubule⁴⁴; the authors concluded that these effects would likely result in changes in heart rate. On the other hand, 24hrs before exposure there was still a trend of increased HRV (SDNN and RMSSD) in the VDD animals when compared with ND even though the HR among the groups was similar. This suggests that other factors may have contributed to the changes in HRV; VDD has been shown to affect blood pressure, which is a known determinant of HRV⁴⁵.

We have shown that heart rate tends to decrease gradually over a 3-4hr air exposure as the animal calms down in the chamber. In the presence of air pollution, heart rate does not decrease as much (i.e. remains elevated)^{23, 46}. In the current study, although SA-PM prevented HR from decreasing in both ND and VDD groups, the effect was significantly less in the VDD mice (i.e. HR of VDD mice was less elevated), which along with the greater increase in RMSSD and HF appears to confirm the shift towards parasympathetic modulation in the VDD animals. This modulation was also reflected in the greater decrease in HR and increase in RMSSD in VDD animals exposed to air. These trends persisted immediately after exposure with HR continuing to decrease over the next 24hrs. Over the same period SDNN and RMSSD continually increased in all groups except ND animals exposed to SA-PM, which consequently had the smallest decrease in HR. It is unclear whether this relatively smaller decrease in HR and increase in HRV in the ND mice exposed to SA-PM reflects increased risk^{47, 48}. These responses are similar to what was observed and reported for SA-PM by Hazari et al. in this issue with the difference in effects due to VDD being evident here. Indeed, this group had a significantly increased number of arrhythmia during exposure but these stopped after it ended. Similar increases in arrhythmia incidence were observed in the other three treated groups, especially VDD mice exposed to air which had a large number of atrial premature beats, but they were not significant. Yet, there is some indication from past studies that VDD is associated with arrhythmia⁴⁹ due to a number of mechanisms, either by activation of the renin-angiotensin-aldosterone (RAS) axis and predisposition to hypertension³, through the enhancement of myocardial oxidative stress^{50, 51}, or by increasing parathyroid hormone (PTH) levels which also affects blood pressure and myocardial contractility^{52, 53}. Whether these conditions existed in our animals is unknown and further does it worsen the response to air pollution likely remains to be clarified.

Assessments of ventilatory function were also performed from the beginning of the diet regimen to the end of the study and revealed some diet-induced effects. Although the animals were randomly assigned to either the normal or deficient diet, the VDD group had a higher breathing frequency than ND before commencing the diets. This difference did not exist at 15 weeks of age. In contrast, VDD animals had a significantly reduced increase in tidal volume over the 12-week diet regimen when compared with ND. Numerous studies have documented the negative effects of VDD on lung development in both humans and rodents^54–56. Furthermore, it appears that VDD causes deficits in lung function that are primarily explained by differences in lung volume and also exacerbates the development of lung COPD-like characteristics (i.e. inflammation) in mice exposed to cigarette smoke. Two important points are relevant here given these conditions: first, it is clear that the dose of SA-PM in VDD animals may have been different from ND animals, and second, it is likely the airway responsiveness of VDD mice to SA-PM was also changed. We cannot address the first point since we were unable to measure ventilatory function during exposure. However, when compared to pre-exposure, VDD animals had a significantly smaller decrease in breathing frequency after exposure than ND and this effect was even greater when VDD and SA-PM were combined. A similar trend was observed in ventilatory timing (penh), which is an indicator of airway irritation, suggesting VDD may have altered the ventilatory responses to the smog. This is relevant to the overall hypothesis of this study because ventilatory patterns not only affect HRV but also can play a role in adverse cardiac events (e.g. arrhythmia) when triggered by airway irritation⁵⁷.

Finally, there were some notable changes in ECG parameters in this study. Although there were no pre-exposure differences in any of the ECG parameters between any of the groups, SA-PM exposure caused a significant increase in PR interval and decrease in QRS complex duration when compared to air; this smog effect was altered by VDD and persisted for 24hrs after exposure only in that group. Even though these changes may just represent fluctuations in the heart rate and not any true electrical disturbances or pathology there is a clear impact of VDD by which the SA-PM-induced effect continued into the next day. In such a situation, if these ECG changes truly were due to an electrical disturbance then it would not be inconceivable for a subsequent adverse cardiac event to be triggered, even if hours after the exposure had ended. In addition, our animals were only kept on the diet for 16 weeks, which may not have been enough time for VDD to cause more tangible effects. For example, chronic changes in blood pressure are connected with vitamin D levels but this was not evaluated in this study. Thus, future experiments will be conducted on animals that are kept on VDD for more than 32 weeks and will include other physiological measures that will help to potentially clarify the ECG phenomenon.

In conclusion, persistent VDD that begins in early-life alters HR, HRV and ventilatory parameters and changes the response to smog. It is still unknown whether these changes were due to a difference in response to particular components, although it appears unlikely because the effect of smog on VDD mice was the same as ND only bigger. VDD mice had less increase in HR and a greater increase in RMSSD and breathing frequency during smog than ND, which was possibly due to the shift in baseline autonomic function. This baseline autonomic imbalance may represent altered homeostasis, which potentially suggests that the body is prone to an adverse response if a subsequent environmental trigger or stressor is encountered. Although this has yet to be proven for VDD, our previous studies demonstrate that autonomic imbalance contributes to triggered cardiac dysfunction after air pollution exposure. This applies to children who are VDD and may be at heightened risk of developing cardiovascular disease later in life and so may also be sensitive to the effects of air pollution. Such effect modification due to nutritional deficiency could have significant relevance to public health and the assessment of toxicological risk.